Kaposi's Sarcoma-Associated Herpesvirus-Encoded La
http://www.100md.com
病菌学杂志 2006年第2期
Department of Microbiology and Tumor Virology Program, Abramson Comprehensive Cancer Center, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania
ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is predominantly associated with three human malignancies, KS, primary effusion lymphoma, and multicentric Castleman's disease. These disorders are linked to genomic instability, known to be a crucial component of the oncogenic process. Latency-associated nuclear antigen (LANA), encoded by open reading frame 73 of the KSHV genome, is a latent protein consistently expressed in all KSHV-associated diseases. LANA is important in viral genome maintenance and is associated with cellular and viral proteins to regulate viral and cellular gene expression. LANA interacts with the tumor suppressor genes p53 and pRb, indicating that LANA may target these proteins and promote oncogenesis. In this study, we generated cell lines which stably expressed LANA to observe the effects of LANA expression on cell phenotype. LANA expression in these stable cell lines showed a dramatic increase in chromosomal instability, indicated by the presence of increased multinucleation, micronuclei, and aberrant centrosomes. In addition, these stable cell lines demonstrated an increased proliferation rate and as well as increased entry into S phase in both stable and transiently transfected LANA-expressing cells. Additionally, p53 transcription and its transactivation activity were suppressed by LANA expression in a dose-dependent manner. LANA may therefore promote chromosomal instability by suppressing the functional activities of p53, thereby facilitating KSHV-mediated pathogenesis and cancer.
INTRODUCTION
Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8, a gamma-2 herpesvirus, was identified by representational differential analysis in a KS biopsy sample from an AIDS patient in 1994 (10). Primarily, KSHV has been strongly associated with KS, primary effusion lymphoma (PEL), and multicentric Castleman's disease (40). Although extended efforts have been taken in studying the development and progression of the diseases, the mechanism of carcinogenesis is still largely unknown. To date, studies suggest that KSHV-associated diseases result from serial genetic alterations driven by ongoing genetic instability, as seen in numerous cancers. For example, recurrent chromosomal abnormalities have been noted in both KSHV-positive PEL patients and cell lines (9, 19, 34, 53). In addition, microsatellite instability, a biomarker of genetic instability, was also detected in a KSHV-positive PEL patient (20). Moreover, KSHV infection induces abnormal mitotic spindles and centrosome duplication, chromosomal misalignment, mitotic bridges, formation of micronuclei, and multinucleation in primary human umbilical vein endothelial cells (42), which indicates that KSHV infection may predispose human cells to chromosomal instability and suggests that genetic instability is likely to be an important contributor leading to the development of KSHV-related diseases.
Typically, KSHV displays two modes of infection: latent infection, during which the viral genome persists in the host cell and no viral progeny are released, and lytic infection, during which the host cell is destroyed and viral progeny are produced. During latent infection, gene expression is limited to a small number of viral genes, which include the latency-associated nuclear antigen (LANA), viral cyclin (v-cyclin), viral Fas-associated death domain interleukin-1L-converting enzyme inhibitory protein (v-FLIP), viral interferon regulatory factor (K10), and kaposin (48). Although lytic infection has been suggested to contribute in part to KSHV-associated pathogenesis (24), latent infection is likely to play a critical role in all KSHV-induced tumors (16, 23, 26, 45).
LANA, encoded by open reading frame 73 (ORF73), is one of the viral genes constitutively expressed during latent infection. LANA is a large nuclear protein (222 to 234 kDa, based on analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) with multifunctional and critical roles in supporting the maintenance and oncogenesis mediated by KSHV. LANA tethers the viral episome to the host genome through its interaction with histone H1 and possibly other cellular proteins, including DEK and methyl-CpG-binding protein (3, 30). It is also important for the maintenance of latency by repressing the transcriptional activity of Rta, a KSHV gene which activates lytic replication (33). LANA activates the transcription factor-4/cyclic AMP response element-binding protein-2 and mSin3A to mediate transcriptional repression and associates with a number of cellular proteins involved in transcriptional regulation, such as CREB-binding protein and RING3 (31, 36). In addition, LANA may modify transcriptional activity by altering the subcellular distribution of GSK-3b, a negative regulator of -catenin (17). LANA can also repress the transcriptional activity of p53 and, furthermore, inhibits the ability of p53 to induce cell death (16). LANA was also found to interact with pRb protein and concurrently stimulates transcription from a cyclin E promoter (43). The findings indicate that LANA may contribute to the oncogenesis of KSHV by deregulating both p53 and pRb tumor suppressor pathways. So far, there are serial lines of evidence which suggested that LANA is a critical protein for oncogenesis. Coexpression of LANA and H-Ras in rat embryonic fibroblasts transforms primary rat cells in vitro (43). LANA also prolongs the life span of human endothelial cells but does not induce the transformation of these cells (52). LANA expression protects lymphoid cells from p16INK4a-induced cell cycle arrest and induces S-phase entry (1). These studies strongly suggest that LANA is a major destabilizer of cell cycle regulation in infected cells and a contributing factor to cell immortalization.
Numerous studies have supported a major role for p53 in the regulation of cell growth and cell death, and recent study provides evidence that p53 also functions in the maintenance of genetic stability (2). Here we show that the long-term expression of LANA promotes chromosomal instability in human cells and that this activity is likely to be through its transcriptional activity, which results in the downregulation of p53 expression.
MATERIALS AND METHODS
Plasmids and antibodies. pA3M-LANA and the pA3M-LANA deletion constructs carrying c-Myc-tagged ORF73, the amino-terminal domain (amino acids 1 to 435), and the carboxyl-terminal domain (amino acids 762 to 1162) under the control of the cytomegalovirus immediate early promoter were constructed by PCR amplification of the ORF, followed by EcoRI and BamHI digestion and insertion into the pA3M vector (23). Red fluorescent protein (RFP)-LANA was constructed by digesting the full-length LANA ORF from the pA3M-LANA plasmid at the EcoRV and HindIII sites and inserting it into the pDsRed1-N1 (Promega, Inc., Madison, WI) vector at the sites of EcoRI (filled in) and HindIII upstream of pDsRed gene.
The p53 reporter plasmid pG13-Luc contains 13 p53-binding sites upstream of the luciferase gene (gift from W. El-Deiry, University of Pennsylvania, Philadelphia) (51) and was constructed by EcoRV insertion of p53-binding sequences into the pGL-2 luciferase reporter plasmid (Promega, Inc., Madison, WI.).
The DO-1 anti-p53 monoclonal antibodies were obtained from Santa Cruz Biotechnologies, Inc. (Santa Cruz, Inc., CA). The 9E10 anti-Myc monoclonal antibody was obtained from the University of Michigan Hybridoma Core Facility. Human KS serum was produced by a Kaposi's sarcoma-positive patient and has cross-reactivity to LANA. The monoclonal antibodies against -tubulin and -tubulin were obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO).
Cell culture and generation of cells stably expressing LANA. The human cervical cancer cell line HeLa and fibroblast cell line Rat1 were grown in Dulbecco's modified Eagle's medium supplemented with antibiotics, L-glutamine, and 5% fetal bovine serum. Cell lines were transfected by electroporation at 210 V and 975 μF as per the manufacturer's instructions. p53–/– and p53+/+ murine embryonic fibroblasts (MEF; gifts from C. B. Thompson, University of Pennsylvania, Philadelphia, PA) (55) were cultured in Dulbecco's modified Eagle's medium supplemented with antibiotics, L-glutamine, and 7% fetal bovine serum. MEF lines were transfected by electroporation at 240 V and 975 μF. The human B-cell line BJAB was grown in RPMI 1640 supplemented with antibiotics, L-glutamine, and 7% fetal bovine serum and transfected by electroporation at 220 V and 975 μF.
To generate cells with stable expression of LANA protein, HeLa, Rat1, and BJAB cells were transfected with the RFP-LANA construct or pDsRed1 vector and subjected to G418 selection (0.4 to 0.8 mg/ml). After an 8-week selection, vector control cells, and cells stably transfected with LANA from 10 independently derived clones were examined for the level of LANA expression through an analysis of its mRNA levels by reverse transcription (RT)-PCR and its protein levels by Western blotting. The change in cell morphology was closely monitored to examine for possible artifacts in the selection process.
To determine the effects of LANA and LANA derivatives on chromosomal instability, the pA3M-LANA and the pA3M-LANA deletion constructs were introduced into Rat1 cells and MEF, respectively, and subjected to G418 (0.4 to 1 mg/ml) for 3 weeks. The change in cell morphology was also closely monitored.
Detection of chromosome instability. The number of cell nuclei was determined by staining cells with the DNA dye 4',6'-diamidino-2-phenylindole (DAPI). HeLa cells, Rat1 cells, and MEF were grown on chamber slides overnight, and BJAB cells were washed with PBS and spread evenly on a slide. The cells were fixed with 1:1 methanol-acetone for 10 min at –20°C and then stained with DAPI at 1 μg/ml for 10 min at room temperature. Slides were examined with an Olympus IX70 fluorescence microscope, and images were captured with a PixelFly digital camera (Cooke, Inc., Auburn Hills, MI). The presence of multinucleation, micronuclei, and chromosomal mitotic bridges was examined after DAPI staining of mitotic cells. To quantify the cells with nuclear changes, at least 200 cells from three randomly selected fields were counted to determine the percentage and to obtain the standard deviation for each sample.
Proliferation assay. A total of 3 x 104 HeLa cells, 4 x 104 Rat1 cells, or 2 x 105 BJAB cells were plated into each well of six-well plates and cultured at 37°C in complete medium. Cells from each well were trypsinized and counted using a hemocytometer and trypan blue exclusion daily for 5 days. Experiments were performed using cells that stably express the LANA protein and were repeated three times.
Cell cycle analysis. To investigate the effect of LANA protein on cell cycle, LANA-expressing cells were subjected to FACScan analysis (BD Biosciences, San Jose, CA) under asynchronized and synchronized conditions. For synchronization, LANA-expressing cells were incubated with medium with 100 ng/ml nocodazole for 18 h. Nocodazole medium was then replaced with normal medium and grew for another 5 h. Cells were harvested by washing once in 1x phosphate-buffered saline and fixing in 70% ethanol. The fixed cells were washed again with PBS twice and treated with RNase A at 37°C for 30 min. Finally, the cells were stained with propidium iodide and incubated in the dark for 60 min or overnight before analysis. The samples were analyzed through flow cytometry using a fluorescence-activated cell sorter manufactured by BD Biosciences (San Jose, CA) and the Cell-Quest program.
p53-responsive transactivation assays. In this study, the transcription activity of p53 was determined by luciferase reporter analysis using the pG13 reporter construct and measuring the luciferase activity (6). Briefly, 10 million cells were transfected with 1 μg of pG13 with increasing amounts of pA3M-LANA. Three micrograms of a green fluorescent protein expression vector was transfected per sample to measure transfection efficiencies. In p53–/– cells, 10 μg of the p53 construct was introduced in addition to LANA and reporter constructs. Cells were harvested 15 h later, washed with phosphate-buffered saline, and lysed in luciferase cell culture lysis buffer (Promega, Inc., Madison, WI). Cellular debris was pelleted, and 30 μl of the supernatant was used for each measurement of luciferase activity with an Opticomp I luminometer (MGM Instruments, Inc., Hamden, CT). Aliquots of cell lysate were saved for confirmation of protein expression by Western blot analysis.
Immunofluorescence assays. HeLa and Rat1 cells grown on chamber slides overnight and BJAB cells were washed with PBS and spread evenly on a slide. The cells were fixed with 1:1 methanol-acetone for 10 min at –20°C, dried, and rehydrated with PBS. For blocking, cells were incubated with PBS containing 3% bovine serum albumin and 1% glycine for 30 min. Cells were then cross-reacted with appropriate antibodies (human KS serum, a 1:500 dilution of anti--tubulin, and a 1:250 dilution of anti--tubulin). Slides were washed three times in phosphate-buffered saline and cross-reacted with a 1:1,000 dilution of goat anti-rabbit or goat anti-mouse immunoglobulin-fluorescein isothiocyanate-conjugated secondary antibodies. Slides were examined with an Olympus IX70 fluorescence microscope, and images were captured with a PixelFly digital camera (Cooke, Inc., Auburn Hills, MI).
Western blot analysis. Electrophoresed proteins were blotted onto 0.45-μm nitrocellulose paper (Osmonics, Inc., Minnetonka, MN) at 100 V for 1 h. Blots were blocked with 5% milk in phosphate-buffered saline and washed three times with TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) before overnight incubation with the rabbit anti-LANA antiserum, the 9E10 anti-Myc hybridoma supernatant, or a 1:500 dilution of the anti-p53 antibody at 4°C. Blots were washed three times with TBST and incubated with a 1:10,000 dilution of the appropriate Alexa Fluor 680 or IRDye800 secondary antibody (Molecular Probes, Eugene, OR). Membranes were scanned with an Odyssey fluorescence scanner (Li-Cor, Lincoln, NE). Densitometric analysis was performed with the Odyssey scanning software.
Real time-PCR analysis of viral and cellular transcription. Total RNA was isolated from cells using TRIZOL reagent (Life Technologies, Gaithersburg, MD). Reverse transcription was carried out with the SuperScript II RNase H-reverse transcriptase (Life Technologies, Gaithersburg, MD). Briefly, five micrograms of total RNA was reverse transcribed by using 200 U of Moloney murine leukemia virus reverse transcriptase in a total volume of 20 μl containing 125 μM deoxynucleoside triphosphate, 20 U of RNasin, and 50 ng of random hexanucleotide primers. After incubation at 25°C for 10 min followed by 50°C for 50 min, the reaction was stopped by heating the mixture to 95°C for 5 min. Quantitative real-time RT-PCR was then performed in a total volume of 25 μl, including 12.5 μl of SYBR green PCR master mix (New England Biolabs, Beverly, MA). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) transcripts in each sample were first amplified as internal controls to normalize mRNA input for LANA amplification. The LANA gene was amplified for 30 cycles (30 s at 94°C, 1 min at 55°C, and 1 min 30 s at 72°C) using the forward primer 5'-ATGTGACTTCGCCAACCGTAG-3' and the reverse primer 5'-TGCTTCTTCTGCAATCTCCG-3'. Amplification products were also resolved in 1.5% agarose gels.
RESULTS
Generation of stable HeLa, Rat1, and BJAB RFP-LANA cell lines. To assess the potential ability of LANA to induce chromosomal instability, cell lines were generated stably expressing the RFP-LANA fusion protein. A cDNA fragment encoding the LANA ORF was cloned into the pDsRed1 plasmid, which consists of a simian virus 40 (SV40) early promoter, a neomycin/kanamycin resistance gene, and a polyadenylation signal from the herpes simplex virus thymidine kinase gene. The signals for the RFP-LANA fusion protein suggest a predominantly nuclear localization compared to that of the parental RFP vector, with signals distributed throughout the cells (Fig. 1A, subpanels a and c). The human cervical cancer cell line HeLa, Rat1 fibroblast line, and the B-cell line BJAB were transfected with the RFP-LANA construct and the pDsRed1 vector as a mock control by electroporation. The RFP-expressing cells were subjected to 400 to 800 μg/ml G418 selection for 8 weeks. To determine whether or not the RFP fluorescence faithfully reflected the actual expression of LANA in the stable cells, HeLa and Rat1 cell lines were grown overnight on chamber slides and fixed with acetone-methanol. BJAB cells were washed with PBS and fixed with acetone-methanol. Cells were then incubated with LANA-specific antiserum for the detection of LANA protein by immunofluorescence. LANA was detected in the RFP-LANA stable cell lines, and the signals colocalized with the RFP signals, suggesting that the RFP-LANA fusion protein was stably expressed in the generated cell lines (Fig. 1B).
Cell lysates prepared from both RFP control cells and RFP-LANA-expressing cells were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis with rabbit polyclonal anti-LANA antiserum. As expected, RFP-LANA expression was detected in samples of RFP-LANA stable cell lines but not from the mock control (Fig. 1C). In addition, mRNA transcription for LANA was detected by RT-PCR (data not shown).
Constitutive expression of LANA protein induces multinucleation, micronuclei, and mitotic bridges in the stable cell lines. To determine the effect of constitutive expression of LANA on cellular activities, three clonal cell lines showing strong expression of RFP-LANA and the pDsRed control vector were stained with DAPI for nuclear analysis. In comparing nuclear morphological changes, the mock control used in our study, the vector pDsRed1, showed little or no chromosomal damage in the expressing cells (Fig. 2A to C). However, the results showed that all three clonal cell lines expressing LANA displayed a dramatic increase in the number of cells exhibiting nuclear pleiomorphism and multinucleated phenotypes. A representative photograph is shown for one cell line in Fig. 2A, and mitotic bridges are shown in cell lines expressing LANA (Fig. 2B). Quantitative analysis of multinucleated cells revealed that in contrast to mock and vector control cell lines, which had less than 5% of the cells with greater than two nuclei or giant nuclei, the LANA-expressing cell lines frequently displayed cells with two or more polarized nuclei. The percentage of these cells showing this phenotype was constantly about 40% (Fig. 2C). Interestingly, the cells with two nuclei increased from 15% in mock control cells to 30 to 40% in the LANA-expressing cells (Fig. 2C). The cells with more than two nuclei, which are reflective of aneuploidy, increased from approximately 4% to 10% (Fig. 2C), and the number of micronuclei was also doubled from that of vector controls to about 10% of the LANA-positive cells (Fig. 2C).
The formation of anaphase bridges reflects the pressure of structural chromosomal abnormalities, mostly as a result of chromosome breakage (21). Previous studies have shown that KSHV can induce structural chromosomal abnormalities in primary human endothelial cells (42). Here, we studied anaphase bridge formation to detect chromatin strings in cells expressing LANA as well as in controls (Fig. 2B). Anaphase bridges were absent in greater than 200 anaphase/telophase Rat1 cells, as determined from multiple fields in mock control keratinocyte populations (Fig. 2B). However, in representative experiments when multiple fields were analyzed, expression of the RFP-LANA fusion protein was found associated with greater than 2% of cells having anaphase bridges (Fig. 2B).
In Rat1 cells, dramatic chromosomal abnormalities were also seen (Fig. 3A). While the number of cells with two nuclei was almost doubled in LANA-expressing cells (Fig. 3B), the cells seen with aneuploid nuclei having greater than two nuclei exhibited a significant increase in the percentage from approximately 0.6% to greater than 5% (Fig. 3C), an increase of about 8- to 10-fold over the level in the control. Notably, the micronuclei presented in LANA-expressing cells were approximately 2%, whereas there were no significant micronuclei seen in the control cell lines (Fig. 3D). In our B-cell lines (BJAB stable cells), the results were also similar to that seen in the HeLa cells. As shown in Fig. 4, the number of cells with multinucleation was almost double in LANA-expressing cells (Fig. 4D and J). However, the cells with micronuclei increased from about 1% in mock control cells to more than 8% in the LANA-expressing cells (Fig. 4G and K).
In order to investigate the role of LANA in primary cells with little no genetic changes, we transfected LANA into wild-type MEF and p53 null MEF and placed them under selection for 24 days. The cells expressing LANA were compared to control cells with respect to changes in nuclear morphology. The results showed that both p53-negative and -positive cell lines expressing LANA displayed a dramatic increase in the number of cells exhibiting the multinucleated nuclear phenotypes (Fig. 4E and F) and micronuclei (Fig. 4H and I). Quantitative analysis of multinucleated cells revealed that in contrast to mock control cells having approximately 10% of their p53-negative cells and 13% of their p53-positive cells containing multiple or giant nuclei, the LANA-expressing MEF frequently displayed cells with two or more polarized nuclei. The percentages of cells showing this phenotype constantly were more than 15% in p53-negative cells and 20% in p53-positive MEF (Fig. 4J). Notably, the number of cells with micronuclei in both types of LANA-expressing cells increased more than five times that in mock control cells (Fig. 4K).
Carboxy-terminal LANA plays a major role in the induction of chromosomal instability. To determine the domain of the LANA protein involved in the induction of instability, pA3M-LANA, the pA3M-LANA deletion constructs carrying the LANA N-terminal domain (amino acids 1 to 435) and C-terminal domain (amino acids 762 to 1162) were introduced into Rat1 cells and p53–/– MEF. After a 3-week selection with G418, cells were stained with DAPI for nuclear analysis.
In Rat1 cells with the mock control vector, about 15% of cells showed the multinucleated phenotype (Fig. 5A and B), while only 0.5% showed micronuclei (Fig. 5A and B). In contrast, the cells with full-length LANA expression displayed both increased multinuclei (more than 25%) and micronuclei (more than 2%) (Fig. 5A and B). Similar effects were also seen in cells with the C-terminal domain located within amino acids 752 to 1162, in that they showed multinuclei at about 23% and micronuclei at about 1.2% (Fig. 5A and B), somewhat less than that seen with full-length LANA. However, no obvious alteration was observed in cells with N-terminal LANA (Fig. 5A and B). The results indicate that C-terminal but not N-terminal LANA is essential for inducing chromosomal instability. Similarly, this trend was consistently seen in p53+/+ MEF but not so in the p53–/– MEF, suggesting a role for p53 regulation by the carboxyl-terminal domain of LANA (Fig. 5, compare panels C and D with E and F).
Constitutive expression of LANA fusion protein induces abnormal formation of spindle poles and centrosomes. The above-described studies demonstrated that LANA-expressing clones exhibited an abnormal increase in the number of multinucleated cells and cells with micronuclei. Therefore, we wanted to determine whether these cells displayed abnormal mitosis activities as indicated by the presence of aberrant numbers of spindle poles and centrosomes. Quantitative scoring of centrosomes with anti--tubulin antibodies in nuclei revealed the presence of an abnormal number of centrosomes in cell lines constitutively expressing LANA compared with cell lines stably expressing only the RFP empty vectors. In the parental Rat1 cell lines with vector alone, greater than 98% of the cells had normal centrosome numbers, with about 2% or less showing abnormality. However, in the RFP-LANA cell lines, greater than 15% of the cells contained more than three centrosomes per cell, an increase in abnormal centrosome activities of seven- to eightfold (Fig. 6A). Failure to control centrosome duplication results in aberrant spindle formation (12). In this experiment, we also analyzed spindle pole abnormalities as detected by immunostaining with anti--tubulin (Fig. 6B). The results showed that in Rat1 cell lines the RFP-LANA-expressing cells contained abnormal spindle pole activities and that greater than 10% of the cells were abnormal, compared to only less than 2% of the control RFP Rat1 cells (Fig. 6B).
LANA expression increases the rate of cell proliferation and entry into S phase. In an effort to determine the effects on cell growth and proliferation due to chromosomal abnormalities in the RFP-LANA-expressing cell lines, the growth rate and cell cycle profile were analyzed for cells stably expressing LANA compared to vector control RFP cell lines from HeLa cell lines and Rat1 cell lines. The results of our studies showed that the LANA-expressing cells proliferated more rapidly in both the HeLa and Rat1 cell lines than in their corresponding vector controls (Fig. 7A and B). The rate was more dramatic in the Rat1 cell lines than in the HeLa cell line, as a reflection of the potential contribution by the human papilloma virus (HPV) antigens present in these cells. However, it is clear that the LANA-expressing cells proliferated more rapidly in both the HeLa and Rat1 cell lines.
To determine possible changes in cell cycle profile due to LANA, the cellular DNA content was measured by propidium iodide staining and flow cytometry (Fig. 7C and D). The results indicted that the LANA-expressing cell lines constantly had 18 to 20% of the cells in S phase compared to approximately 5% of S-phase cells in vector control cell lines. The difference reflects a higher cell proliferation rate and supports a previous study which suggested that LANA can stimulate S-phase entry as monitored by bromodeoxyuridine incorporation (18). In addition, the number of LANA-expressing cells in G2/M phase and cells with more than 4N DNA was typically higher, 21%, than that in vector control cells, with about 15% confined (Fig. 7, compare panels C and D). In contrast, the number of cells with LANA in G1 phase was decreased from greater than 75% in the vector control to less than 50% in the LANA-expressing cells. These results indicated that the expression of LANA induced rapid cell proliferation and that this increase was likely related to increased chromosomal aberrations.
LANA expression leads to the downregulation of p53 levels. LANA has been shown to repress the transactivation activity of p53 (16, 28). In this study, p53 activity was found to be partially neutralized by LANA expression in both stably and transiently expressing cells. In the cells stably expressing LANA, p53 transcription activity was determined by the detection of luciferase activity from a luciferase reporter construct, pG13-Luc, containing a multimerized p53 consensus-binding site (5). This construct was transfected into BJAB cells with stable LANA expression. Transfection efficiency was monitored by cotransfection with the pEGFP vector, and each transfection was done in triplicate for four LANA-expressing clones (Fig. 8A). The transactivation activity of p53 was normalized to 1.0 and compared to the results for LANA-expressing cell lines. The results show that LANA expression suppressed p53 transactivation activity by approximately 50% in the four cell lines analyzed (Fig. 8A). This suggested that LANA may directly affect p53 activity, but there was the possibility that p53 levels were also depressed in these cells. Western blot analysis of lysates from these cell lines showed a definite reduction in levels of p53 protein compared to that in the vector control cell line (Fig. 8A). In p53–/– MEF, we introduced p53 as well as an increased amount of the LANA construct and pG13-Luc reporter plasmid. The transactivation activity of p53 in mock control (no p53 transfection) cells was normalized to 1.0 (Fig. 8B). The transactivation activity of p53 was determined to be over 18 times higher in cells transfected with p53 (Fig. 8B). Moreover, LANA expression suppressed p53 transactivation activity, and the decrease of p53 activity was dose responsive to the increased level of LANA input (Fig. 8B). Therefore, LANA may regulate levels of p53 transcription from the endogenous promoter or mediate the degradation of the p53 protein, which leads to reduced levels in stable LANA lines.
To directly determine whether LANA can repress p53 expression at the level of transcription or posttranscriptionally, a titration experiment was performed in which BJAB cells were cotransfected with an increased amount of a LANA-expressing construct with a steady amount of reporter pG13-luc. The results showed that an increase in amounts of LANA resulted in decreased levels of p53 transcription activities as determined by reporter assay in a dose-response fashion (Fig. 9A). The levels of endogenous p53 transcripts were also reduced proportionally to input LANA expression (Fig. 9B and C). These results also correlated nicely with the levels of p53 protein as shown by a corresponding reduction in p53 signal by Western blot analysis as the amount of LANA was increased from a heterologous system (Fig. 9D and E). Thus, these results suggest that LANA can repress p53 transactivation activities, at least in part by repressing its endogenous promoter, thus resulting in decreased p53 transcript levels.
DISCUSSION
The generation of cell lines stably expressing the KSHV-encoded LANA in this study allowed us to demonstrate for the first time that LANA can induce chromosomal instability in multiple cell types. The observation that dramatic rises in multinucleation, numbers of micronuclei, and abnormal levels of centrosomes and spindle poles as well as increased proliferation in stable LANA cell lines strongly suggests that constitutive expression of LANA during latent infection may have a predominant role in driving the oncogenic process mediated by KSHV.
Chromosomal instability, a particular hallmark of human cancer, has been considered an adaptive response of cancer cells to environmental pressure (27). Chromosomal instability has been typically defined as an accelerated rate of gains and losses of whole or large portions of chromosomes in the context of continuous cell growth (44). In most cases, chromosomal instability results from numerous chromosomal alterations, as well as additional structural alterations. Defects in chromosome segregation, DNA damage repairing, telomere stability, and cell cycle disturbance are some of the major cellular mechanisms that can contribute to the induction of chromosomal instability (22). Loss of p53 function is a frequent and important event in the genesis and progression of many human malignancies, and a number of studies have suggested that p53-mediated cell cycle control plays a critical role in the preservation of chromosomal stability. Intact p53 responses typically impose barriers to cells with chromosome alterations by stimulating G1/S or G2/M checkpoint responses, mitotic catastrophe, and apoptotic cell death (13). In contrast, cells with an inactivated p53 response may overcome these checkpoints and maintain proliferation regardless of the chromosomal aberrations (54). This is confirmed by the observation that cells lacking p53 or having altered p53 activities have an increased ability to reenter the cell cycle following prolonged spindle disruption and can therefore initiate another round of DNA replication (7, 39, 47).
In the context of gammaherpesvirus oncogenesis, chromosomal instability has been seen in KSHV-associated malignancies; this instability is most likely mediated by one or more of its latent genes. For example, v-cyclin, one of the latently expressed genes, has been shown to block cytokinesis and drive DNA replication in human and mouse primary cells, thus stimulating polyploidy (49, 50). The abnormality triggers p53-dependent apoptosis and growth arrest, and its tumorigenesis is restrained in a p53-dependent manner (49). This is further supported by the fact that the aneuploid population survived and expanded both in vitro and in vivo in the absence of p53, leading to an oncogenic phenotype (49, 50). Therefore, mechanisms for overcoming the p53-dependent checkpoints are likely to be crucial for tumor development in KSHV-associated cancers. LANA has been shown to suppress the transactivation activity of p53 and to protect against p53-mediated cell death and is resistant to p53-dependent apoptosis but not p53-independent apoptosis (16). Elucidating the effect of constitutive LANA expression on p53 transactivation activity and chromosomal integrity will be important and will contribute to understanding the oncogenic properties of KSHV.
To provide insights into LANA's effect on inactivation of p53, this study demonstrated that LANA can induce chromosomal instability by repressing the transcription of p53 from its endogenous promoter. In multiple cell types with different backgrounds where stable cell lines were generated, aneuploidy and defects in spindle poles and centrosomes were consistently increased. These aberrations were more significant in the rat fibroblast cell line Rat1 and murine embryonic fibroblasts with wild-type or null p53 than those seen in HeLa cells. HeLa is a cervical-cancer cell line with high-risk HPV infection and consistent E6 and E7 expression. While high-risk HPV E6 protein disrupts the p53 protein by promoting p53 ubiquitin-mediated degradation, E7 protein disrupts the pRb pathway (15, 46). Previous studies showed that the E6 and E7 proteins cooperate to induce aneuploidy and chromosomal instability (14). In HeLa cells expressing LANA, a further decrease in p53 levels was consistently observed, and these results were also supported with the Rat1 cell line and MEF, suggesting a major contribution by LANA to p53 levels. The increase of aneuploidy was also corroborated by DNA content analysis, which showed that in the LANA-expressing cells, the DNA content in cells with greater than 4N was almost doubled, supporting the chromosomal changes in morphology seen with nuclear staining.
Studies have shown that the amino terminus of LANA mediates chromatin binding and that the carboxyl terminus mediates binding to a 13-bp sequence in the terminal repeats of the viral episome, anchoring the viral episome to the host chromosome (4, 11). The p53-interacting region has been mapped to the C terminus of the LANA protein (6). The N-terminal domain of LANA can neither bind p53 nor antagonize the transactivation activity of p53 (16). The C-terminal domain of the derivative LANA construct used in this study has been shown to retain the ability to bind p53 and downregulate p53 transactivity (6). Our results showed that the C terminus of LANA plays a major role in the induction of chromosomal instability, suggesting that the C terminus is in large part responsible for inducing these abnormalities, which are closely related to its ability to counteract p53 transactivation.
In the LANA-positive cell lines with chromosomal instability, p53 transactivation activity was downregulated. These results are consistent with previous microarray results which showed low p53 transcript levels in LANA-expressing cells (45). p53 function has been determined to be regulated in multiple ways. Virus-encoded oncoproteins (SV40 large T antigen, adenovirus E1B, HPV E6, and hepatitis B virus X) as well as the cellular oncoprotein Mdm-2 have been known to inhibit p53 function via physical interaction, abrogating the specific DNA binding or transactivation activity of p53. This sequesters p53 in the cytoplasm or accelerates its degradation (38). In KSHV, LANA was colocalized with p53 in tissue from KS patients, PEL cells, and human KSHV-associated solid lymphoma (25, 28). In addition, physical interaction between LANA and p53 was demonstrated (6). Furthermore, the region of p53 targeted by LANA and ORF73 from herpesvirus saimiri was mapped to the domain required for tetramerization but does not affect tetramerization (6). Thus, this study suggests another mechanism for p53 regulation, in addition to a physical interaction which involves transcription downregulation.
The DNA content analysis also confirms that LANA promotes the proliferation rate by stimulating S-phase entry (52). This agrees with the results of a previous study showing that LANA can inhibit pRb function. The tumor suppressor pRb sequesters E2F, a transcription factor required for G1/S transition (41). By inhibition of pRb, LANA promotes S-phase entry and prolongs the life span of human primary endothelial cells. The inhibition of both pRb and p53 enables LANA-expressing cells to circumvent the G1/S checkpoint in addition to the apoptotic pathway, thus contributing to oncogenesis. In endothelial cells, LANA expression can be detected very early after infection (29, 32). It is tempting to speculate that LANA inhibits pRb and helps to create a cellular environment supportive of DNA replication and susceptible to the establishment of viral latency. Such a high DNA replication demand, as well as spontaneous errors in host genome replication, is likely to drive an increase in errors during chromosome segregation. Supportively, LANA then suppresses p53 function, thus allowing the cells with mitotic aberrations to survive, resulting in the accumulation of chromosomal instability.
This study also may provide clues as to why LANA is incapable of transforming endothelial cells. Several lines of evidence indicate that the inactivation of p53 function alone is not sufficient to cause chromosomal instability. Loss of p53 does not necessarily lead to aneuploidy, as targeted disruption of p53 in either diploid tumor cells or normal human fibroblasts is not typically associated with aneuploidy even after several population doublings (8). Moreover, several cancer cell lines with p53 mutations are diploid and chromosomally stable (35). Mice with heterozygous p53 burdened with a mutant form of SV40 large T antigen that retained the ability to inactivate pRb but not p53 developed tumors and showed a diploid karyotype (37). This is also supported by results which showed that in vitro-generated neoplastic cells expressing human TERT, H-ras, and SV40 oncogenes are hyperproliferative but genomically stable despite inactivation of p53 by the SV40 large T antigen. Finally, a recent study showed that cells with partial impairment of p53 function retained normal centrosome numbers as wild-type cells (35). However, when the cells were exposed to DNA damage, they developed centrosome amplification, whereas cells carrying wild-type p53 maintained normal centrosome numbers (35). Hence, p53 inactivation may not represent the only contributor to chromosomal instability but may rather facilitate chromosomal instability in cooperation with additional cellular insults. Notably, our study showed that the transactivation activity of p53 is only partially suppressed. Additional steps may be involved in the development of chromosomal instability. Chromosome aberration may initially be stimulated by cellular insults, and since the cell lines used in this study are established cell lines, it is tempting to speculate that these insults may occur more frequently than in primary cells. In addition, replication errors may also be driven by the increased replication rate induced by LANA. These events are then followed by LANA suppression of p53 transcription activity, allowing for the accumulation of aberrations and the facilitation of chromosomal instability in these cells.
In the context of KSHV pathology, KSHV infection, establishment of latency, and the expression of latent genes, such as v-cyclin and v-FLIP, LANA proteins are important in driving oncogenesis (Fig. 10). The expression of these latent genes drives cells to undergo rapid proliferation, replication, and abortive cytokinesis and adapt from the defect in cytokinesis. Accompanied by other spontaneous cellular errors, these aberrations can trigger the activation of p53 followed by concurrent growth arrest and apoptosis. However, suppression of p53 transcript levels by LANA expression leads to a failure to monitor the generated aberrations by p53-dependent checkpoints, thus resulting in the formation of a polyploid/aneuploid population of cells with amplified numbers of centrosomes. The accumulated abnormalities lead to chromosomal instability and KSHV-associated cancers.
ACKNOWLEDGMENTS
This work was supported by grants from the Leukemia and Lymphoma Society of America and by public health service grants NCI CA072510 and CA091792 and NIDCR DE01436 (to E.S.R.). E.S.R. is a scholar of the Leukemia and Lymphoma Society of America.
We also thank Jianping Wang for her technical assistance and other members of the Robertson lab, especially Subash Verma, for helpful suggestions.
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ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is predominantly associated with three human malignancies, KS, primary effusion lymphoma, and multicentric Castleman's disease. These disorders are linked to genomic instability, known to be a crucial component of the oncogenic process. Latency-associated nuclear antigen (LANA), encoded by open reading frame 73 of the KSHV genome, is a latent protein consistently expressed in all KSHV-associated diseases. LANA is important in viral genome maintenance and is associated with cellular and viral proteins to regulate viral and cellular gene expression. LANA interacts with the tumor suppressor genes p53 and pRb, indicating that LANA may target these proteins and promote oncogenesis. In this study, we generated cell lines which stably expressed LANA to observe the effects of LANA expression on cell phenotype. LANA expression in these stable cell lines showed a dramatic increase in chromosomal instability, indicated by the presence of increased multinucleation, micronuclei, and aberrant centrosomes. In addition, these stable cell lines demonstrated an increased proliferation rate and as well as increased entry into S phase in both stable and transiently transfected LANA-expressing cells. Additionally, p53 transcription and its transactivation activity were suppressed by LANA expression in a dose-dependent manner. LANA may therefore promote chromosomal instability by suppressing the functional activities of p53, thereby facilitating KSHV-mediated pathogenesis and cancer.
INTRODUCTION
Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8, a gamma-2 herpesvirus, was identified by representational differential analysis in a KS biopsy sample from an AIDS patient in 1994 (10). Primarily, KSHV has been strongly associated with KS, primary effusion lymphoma (PEL), and multicentric Castleman's disease (40). Although extended efforts have been taken in studying the development and progression of the diseases, the mechanism of carcinogenesis is still largely unknown. To date, studies suggest that KSHV-associated diseases result from serial genetic alterations driven by ongoing genetic instability, as seen in numerous cancers. For example, recurrent chromosomal abnormalities have been noted in both KSHV-positive PEL patients and cell lines (9, 19, 34, 53). In addition, microsatellite instability, a biomarker of genetic instability, was also detected in a KSHV-positive PEL patient (20). Moreover, KSHV infection induces abnormal mitotic spindles and centrosome duplication, chromosomal misalignment, mitotic bridges, formation of micronuclei, and multinucleation in primary human umbilical vein endothelial cells (42), which indicates that KSHV infection may predispose human cells to chromosomal instability and suggests that genetic instability is likely to be an important contributor leading to the development of KSHV-related diseases.
Typically, KSHV displays two modes of infection: latent infection, during which the viral genome persists in the host cell and no viral progeny are released, and lytic infection, during which the host cell is destroyed and viral progeny are produced. During latent infection, gene expression is limited to a small number of viral genes, which include the latency-associated nuclear antigen (LANA), viral cyclin (v-cyclin), viral Fas-associated death domain interleukin-1L-converting enzyme inhibitory protein (v-FLIP), viral interferon regulatory factor (K10), and kaposin (48). Although lytic infection has been suggested to contribute in part to KSHV-associated pathogenesis (24), latent infection is likely to play a critical role in all KSHV-induced tumors (16, 23, 26, 45).
LANA, encoded by open reading frame 73 (ORF73), is one of the viral genes constitutively expressed during latent infection. LANA is a large nuclear protein (222 to 234 kDa, based on analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) with multifunctional and critical roles in supporting the maintenance and oncogenesis mediated by KSHV. LANA tethers the viral episome to the host genome through its interaction with histone H1 and possibly other cellular proteins, including DEK and methyl-CpG-binding protein (3, 30). It is also important for the maintenance of latency by repressing the transcriptional activity of Rta, a KSHV gene which activates lytic replication (33). LANA activates the transcription factor-4/cyclic AMP response element-binding protein-2 and mSin3A to mediate transcriptional repression and associates with a number of cellular proteins involved in transcriptional regulation, such as CREB-binding protein and RING3 (31, 36). In addition, LANA may modify transcriptional activity by altering the subcellular distribution of GSK-3b, a negative regulator of -catenin (17). LANA can also repress the transcriptional activity of p53 and, furthermore, inhibits the ability of p53 to induce cell death (16). LANA was also found to interact with pRb protein and concurrently stimulates transcription from a cyclin E promoter (43). The findings indicate that LANA may contribute to the oncogenesis of KSHV by deregulating both p53 and pRb tumor suppressor pathways. So far, there are serial lines of evidence which suggested that LANA is a critical protein for oncogenesis. Coexpression of LANA and H-Ras in rat embryonic fibroblasts transforms primary rat cells in vitro (43). LANA also prolongs the life span of human endothelial cells but does not induce the transformation of these cells (52). LANA expression protects lymphoid cells from p16INK4a-induced cell cycle arrest and induces S-phase entry (1). These studies strongly suggest that LANA is a major destabilizer of cell cycle regulation in infected cells and a contributing factor to cell immortalization.
Numerous studies have supported a major role for p53 in the regulation of cell growth and cell death, and recent study provides evidence that p53 also functions in the maintenance of genetic stability (2). Here we show that the long-term expression of LANA promotes chromosomal instability in human cells and that this activity is likely to be through its transcriptional activity, which results in the downregulation of p53 expression.
MATERIALS AND METHODS
Plasmids and antibodies. pA3M-LANA and the pA3M-LANA deletion constructs carrying c-Myc-tagged ORF73, the amino-terminal domain (amino acids 1 to 435), and the carboxyl-terminal domain (amino acids 762 to 1162) under the control of the cytomegalovirus immediate early promoter were constructed by PCR amplification of the ORF, followed by EcoRI and BamHI digestion and insertion into the pA3M vector (23). Red fluorescent protein (RFP)-LANA was constructed by digesting the full-length LANA ORF from the pA3M-LANA plasmid at the EcoRV and HindIII sites and inserting it into the pDsRed1-N1 (Promega, Inc., Madison, WI) vector at the sites of EcoRI (filled in) and HindIII upstream of pDsRed gene.
The p53 reporter plasmid pG13-Luc contains 13 p53-binding sites upstream of the luciferase gene (gift from W. El-Deiry, University of Pennsylvania, Philadelphia) (51) and was constructed by EcoRV insertion of p53-binding sequences into the pGL-2 luciferase reporter plasmid (Promega, Inc., Madison, WI.).
The DO-1 anti-p53 monoclonal antibodies were obtained from Santa Cruz Biotechnologies, Inc. (Santa Cruz, Inc., CA). The 9E10 anti-Myc monoclonal antibody was obtained from the University of Michigan Hybridoma Core Facility. Human KS serum was produced by a Kaposi's sarcoma-positive patient and has cross-reactivity to LANA. The monoclonal antibodies against -tubulin and -tubulin were obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO).
Cell culture and generation of cells stably expressing LANA. The human cervical cancer cell line HeLa and fibroblast cell line Rat1 were grown in Dulbecco's modified Eagle's medium supplemented with antibiotics, L-glutamine, and 5% fetal bovine serum. Cell lines were transfected by electroporation at 210 V and 975 μF as per the manufacturer's instructions. p53–/– and p53+/+ murine embryonic fibroblasts (MEF; gifts from C. B. Thompson, University of Pennsylvania, Philadelphia, PA) (55) were cultured in Dulbecco's modified Eagle's medium supplemented with antibiotics, L-glutamine, and 7% fetal bovine serum. MEF lines were transfected by electroporation at 240 V and 975 μF. The human B-cell line BJAB was grown in RPMI 1640 supplemented with antibiotics, L-glutamine, and 7% fetal bovine serum and transfected by electroporation at 220 V and 975 μF.
To generate cells with stable expression of LANA protein, HeLa, Rat1, and BJAB cells were transfected with the RFP-LANA construct or pDsRed1 vector and subjected to G418 selection (0.4 to 0.8 mg/ml). After an 8-week selection, vector control cells, and cells stably transfected with LANA from 10 independently derived clones were examined for the level of LANA expression through an analysis of its mRNA levels by reverse transcription (RT)-PCR and its protein levels by Western blotting. The change in cell morphology was closely monitored to examine for possible artifacts in the selection process.
To determine the effects of LANA and LANA derivatives on chromosomal instability, the pA3M-LANA and the pA3M-LANA deletion constructs were introduced into Rat1 cells and MEF, respectively, and subjected to G418 (0.4 to 1 mg/ml) for 3 weeks. The change in cell morphology was also closely monitored.
Detection of chromosome instability. The number of cell nuclei was determined by staining cells with the DNA dye 4',6'-diamidino-2-phenylindole (DAPI). HeLa cells, Rat1 cells, and MEF were grown on chamber slides overnight, and BJAB cells were washed with PBS and spread evenly on a slide. The cells were fixed with 1:1 methanol-acetone for 10 min at –20°C and then stained with DAPI at 1 μg/ml for 10 min at room temperature. Slides were examined with an Olympus IX70 fluorescence microscope, and images were captured with a PixelFly digital camera (Cooke, Inc., Auburn Hills, MI). The presence of multinucleation, micronuclei, and chromosomal mitotic bridges was examined after DAPI staining of mitotic cells. To quantify the cells with nuclear changes, at least 200 cells from three randomly selected fields were counted to determine the percentage and to obtain the standard deviation for each sample.
Proliferation assay. A total of 3 x 104 HeLa cells, 4 x 104 Rat1 cells, or 2 x 105 BJAB cells were plated into each well of six-well plates and cultured at 37°C in complete medium. Cells from each well were trypsinized and counted using a hemocytometer and trypan blue exclusion daily for 5 days. Experiments were performed using cells that stably express the LANA protein and were repeated three times.
Cell cycle analysis. To investigate the effect of LANA protein on cell cycle, LANA-expressing cells were subjected to FACScan analysis (BD Biosciences, San Jose, CA) under asynchronized and synchronized conditions. For synchronization, LANA-expressing cells were incubated with medium with 100 ng/ml nocodazole for 18 h. Nocodazole medium was then replaced with normal medium and grew for another 5 h. Cells were harvested by washing once in 1x phosphate-buffered saline and fixing in 70% ethanol. The fixed cells were washed again with PBS twice and treated with RNase A at 37°C for 30 min. Finally, the cells were stained with propidium iodide and incubated in the dark for 60 min or overnight before analysis. The samples were analyzed through flow cytometry using a fluorescence-activated cell sorter manufactured by BD Biosciences (San Jose, CA) and the Cell-Quest program.
p53-responsive transactivation assays. In this study, the transcription activity of p53 was determined by luciferase reporter analysis using the pG13 reporter construct and measuring the luciferase activity (6). Briefly, 10 million cells were transfected with 1 μg of pG13 with increasing amounts of pA3M-LANA. Three micrograms of a green fluorescent protein expression vector was transfected per sample to measure transfection efficiencies. In p53–/– cells, 10 μg of the p53 construct was introduced in addition to LANA and reporter constructs. Cells were harvested 15 h later, washed with phosphate-buffered saline, and lysed in luciferase cell culture lysis buffer (Promega, Inc., Madison, WI). Cellular debris was pelleted, and 30 μl of the supernatant was used for each measurement of luciferase activity with an Opticomp I luminometer (MGM Instruments, Inc., Hamden, CT). Aliquots of cell lysate were saved for confirmation of protein expression by Western blot analysis.
Immunofluorescence assays. HeLa and Rat1 cells grown on chamber slides overnight and BJAB cells were washed with PBS and spread evenly on a slide. The cells were fixed with 1:1 methanol-acetone for 10 min at –20°C, dried, and rehydrated with PBS. For blocking, cells were incubated with PBS containing 3% bovine serum albumin and 1% glycine for 30 min. Cells were then cross-reacted with appropriate antibodies (human KS serum, a 1:500 dilution of anti--tubulin, and a 1:250 dilution of anti--tubulin). Slides were washed three times in phosphate-buffered saline and cross-reacted with a 1:1,000 dilution of goat anti-rabbit or goat anti-mouse immunoglobulin-fluorescein isothiocyanate-conjugated secondary antibodies. Slides were examined with an Olympus IX70 fluorescence microscope, and images were captured with a PixelFly digital camera (Cooke, Inc., Auburn Hills, MI).
Western blot analysis. Electrophoresed proteins were blotted onto 0.45-μm nitrocellulose paper (Osmonics, Inc., Minnetonka, MN) at 100 V for 1 h. Blots were blocked with 5% milk in phosphate-buffered saline and washed three times with TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) before overnight incubation with the rabbit anti-LANA antiserum, the 9E10 anti-Myc hybridoma supernatant, or a 1:500 dilution of the anti-p53 antibody at 4°C. Blots were washed three times with TBST and incubated with a 1:10,000 dilution of the appropriate Alexa Fluor 680 or IRDye800 secondary antibody (Molecular Probes, Eugene, OR). Membranes were scanned with an Odyssey fluorescence scanner (Li-Cor, Lincoln, NE). Densitometric analysis was performed with the Odyssey scanning software.
Real time-PCR analysis of viral and cellular transcription. Total RNA was isolated from cells using TRIZOL reagent (Life Technologies, Gaithersburg, MD). Reverse transcription was carried out with the SuperScript II RNase H-reverse transcriptase (Life Technologies, Gaithersburg, MD). Briefly, five micrograms of total RNA was reverse transcribed by using 200 U of Moloney murine leukemia virus reverse transcriptase in a total volume of 20 μl containing 125 μM deoxynucleoside triphosphate, 20 U of RNasin, and 50 ng of random hexanucleotide primers. After incubation at 25°C for 10 min followed by 50°C for 50 min, the reaction was stopped by heating the mixture to 95°C for 5 min. Quantitative real-time RT-PCR was then performed in a total volume of 25 μl, including 12.5 μl of SYBR green PCR master mix (New England Biolabs, Beverly, MA). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) transcripts in each sample were first amplified as internal controls to normalize mRNA input for LANA amplification. The LANA gene was amplified for 30 cycles (30 s at 94°C, 1 min at 55°C, and 1 min 30 s at 72°C) using the forward primer 5'-ATGTGACTTCGCCAACCGTAG-3' and the reverse primer 5'-TGCTTCTTCTGCAATCTCCG-3'. Amplification products were also resolved in 1.5% agarose gels.
RESULTS
Generation of stable HeLa, Rat1, and BJAB RFP-LANA cell lines. To assess the potential ability of LANA to induce chromosomal instability, cell lines were generated stably expressing the RFP-LANA fusion protein. A cDNA fragment encoding the LANA ORF was cloned into the pDsRed1 plasmid, which consists of a simian virus 40 (SV40) early promoter, a neomycin/kanamycin resistance gene, and a polyadenylation signal from the herpes simplex virus thymidine kinase gene. The signals for the RFP-LANA fusion protein suggest a predominantly nuclear localization compared to that of the parental RFP vector, with signals distributed throughout the cells (Fig. 1A, subpanels a and c). The human cervical cancer cell line HeLa, Rat1 fibroblast line, and the B-cell line BJAB were transfected with the RFP-LANA construct and the pDsRed1 vector as a mock control by electroporation. The RFP-expressing cells were subjected to 400 to 800 μg/ml G418 selection for 8 weeks. To determine whether or not the RFP fluorescence faithfully reflected the actual expression of LANA in the stable cells, HeLa and Rat1 cell lines were grown overnight on chamber slides and fixed with acetone-methanol. BJAB cells were washed with PBS and fixed with acetone-methanol. Cells were then incubated with LANA-specific antiserum for the detection of LANA protein by immunofluorescence. LANA was detected in the RFP-LANA stable cell lines, and the signals colocalized with the RFP signals, suggesting that the RFP-LANA fusion protein was stably expressed in the generated cell lines (Fig. 1B).
Cell lysates prepared from both RFP control cells and RFP-LANA-expressing cells were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis with rabbit polyclonal anti-LANA antiserum. As expected, RFP-LANA expression was detected in samples of RFP-LANA stable cell lines but not from the mock control (Fig. 1C). In addition, mRNA transcription for LANA was detected by RT-PCR (data not shown).
Constitutive expression of LANA protein induces multinucleation, micronuclei, and mitotic bridges in the stable cell lines. To determine the effect of constitutive expression of LANA on cellular activities, three clonal cell lines showing strong expression of RFP-LANA and the pDsRed control vector were stained with DAPI for nuclear analysis. In comparing nuclear morphological changes, the mock control used in our study, the vector pDsRed1, showed little or no chromosomal damage in the expressing cells (Fig. 2A to C). However, the results showed that all three clonal cell lines expressing LANA displayed a dramatic increase in the number of cells exhibiting nuclear pleiomorphism and multinucleated phenotypes. A representative photograph is shown for one cell line in Fig. 2A, and mitotic bridges are shown in cell lines expressing LANA (Fig. 2B). Quantitative analysis of multinucleated cells revealed that in contrast to mock and vector control cell lines, which had less than 5% of the cells with greater than two nuclei or giant nuclei, the LANA-expressing cell lines frequently displayed cells with two or more polarized nuclei. The percentage of these cells showing this phenotype was constantly about 40% (Fig. 2C). Interestingly, the cells with two nuclei increased from 15% in mock control cells to 30 to 40% in the LANA-expressing cells (Fig. 2C). The cells with more than two nuclei, which are reflective of aneuploidy, increased from approximately 4% to 10% (Fig. 2C), and the number of micronuclei was also doubled from that of vector controls to about 10% of the LANA-positive cells (Fig. 2C).
The formation of anaphase bridges reflects the pressure of structural chromosomal abnormalities, mostly as a result of chromosome breakage (21). Previous studies have shown that KSHV can induce structural chromosomal abnormalities in primary human endothelial cells (42). Here, we studied anaphase bridge formation to detect chromatin strings in cells expressing LANA as well as in controls (Fig. 2B). Anaphase bridges were absent in greater than 200 anaphase/telophase Rat1 cells, as determined from multiple fields in mock control keratinocyte populations (Fig. 2B). However, in representative experiments when multiple fields were analyzed, expression of the RFP-LANA fusion protein was found associated with greater than 2% of cells having anaphase bridges (Fig. 2B).
In Rat1 cells, dramatic chromosomal abnormalities were also seen (Fig. 3A). While the number of cells with two nuclei was almost doubled in LANA-expressing cells (Fig. 3B), the cells seen with aneuploid nuclei having greater than two nuclei exhibited a significant increase in the percentage from approximately 0.6% to greater than 5% (Fig. 3C), an increase of about 8- to 10-fold over the level in the control. Notably, the micronuclei presented in LANA-expressing cells were approximately 2%, whereas there were no significant micronuclei seen in the control cell lines (Fig. 3D). In our B-cell lines (BJAB stable cells), the results were also similar to that seen in the HeLa cells. As shown in Fig. 4, the number of cells with multinucleation was almost double in LANA-expressing cells (Fig. 4D and J). However, the cells with micronuclei increased from about 1% in mock control cells to more than 8% in the LANA-expressing cells (Fig. 4G and K).
In order to investigate the role of LANA in primary cells with little no genetic changes, we transfected LANA into wild-type MEF and p53 null MEF and placed them under selection for 24 days. The cells expressing LANA were compared to control cells with respect to changes in nuclear morphology. The results showed that both p53-negative and -positive cell lines expressing LANA displayed a dramatic increase in the number of cells exhibiting the multinucleated nuclear phenotypes (Fig. 4E and F) and micronuclei (Fig. 4H and I). Quantitative analysis of multinucleated cells revealed that in contrast to mock control cells having approximately 10% of their p53-negative cells and 13% of their p53-positive cells containing multiple or giant nuclei, the LANA-expressing MEF frequently displayed cells with two or more polarized nuclei. The percentages of cells showing this phenotype constantly were more than 15% in p53-negative cells and 20% in p53-positive MEF (Fig. 4J). Notably, the number of cells with micronuclei in both types of LANA-expressing cells increased more than five times that in mock control cells (Fig. 4K).
Carboxy-terminal LANA plays a major role in the induction of chromosomal instability. To determine the domain of the LANA protein involved in the induction of instability, pA3M-LANA, the pA3M-LANA deletion constructs carrying the LANA N-terminal domain (amino acids 1 to 435) and C-terminal domain (amino acids 762 to 1162) were introduced into Rat1 cells and p53–/– MEF. After a 3-week selection with G418, cells were stained with DAPI for nuclear analysis.
In Rat1 cells with the mock control vector, about 15% of cells showed the multinucleated phenotype (Fig. 5A and B), while only 0.5% showed micronuclei (Fig. 5A and B). In contrast, the cells with full-length LANA expression displayed both increased multinuclei (more than 25%) and micronuclei (more than 2%) (Fig. 5A and B). Similar effects were also seen in cells with the C-terminal domain located within amino acids 752 to 1162, in that they showed multinuclei at about 23% and micronuclei at about 1.2% (Fig. 5A and B), somewhat less than that seen with full-length LANA. However, no obvious alteration was observed in cells with N-terminal LANA (Fig. 5A and B). The results indicate that C-terminal but not N-terminal LANA is essential for inducing chromosomal instability. Similarly, this trend was consistently seen in p53+/+ MEF but not so in the p53–/– MEF, suggesting a role for p53 regulation by the carboxyl-terminal domain of LANA (Fig. 5, compare panels C and D with E and F).
Constitutive expression of LANA fusion protein induces abnormal formation of spindle poles and centrosomes. The above-described studies demonstrated that LANA-expressing clones exhibited an abnormal increase in the number of multinucleated cells and cells with micronuclei. Therefore, we wanted to determine whether these cells displayed abnormal mitosis activities as indicated by the presence of aberrant numbers of spindle poles and centrosomes. Quantitative scoring of centrosomes with anti--tubulin antibodies in nuclei revealed the presence of an abnormal number of centrosomes in cell lines constitutively expressing LANA compared with cell lines stably expressing only the RFP empty vectors. In the parental Rat1 cell lines with vector alone, greater than 98% of the cells had normal centrosome numbers, with about 2% or less showing abnormality. However, in the RFP-LANA cell lines, greater than 15% of the cells contained more than three centrosomes per cell, an increase in abnormal centrosome activities of seven- to eightfold (Fig. 6A). Failure to control centrosome duplication results in aberrant spindle formation (12). In this experiment, we also analyzed spindle pole abnormalities as detected by immunostaining with anti--tubulin (Fig. 6B). The results showed that in Rat1 cell lines the RFP-LANA-expressing cells contained abnormal spindle pole activities and that greater than 10% of the cells were abnormal, compared to only less than 2% of the control RFP Rat1 cells (Fig. 6B).
LANA expression increases the rate of cell proliferation and entry into S phase. In an effort to determine the effects on cell growth and proliferation due to chromosomal abnormalities in the RFP-LANA-expressing cell lines, the growth rate and cell cycle profile were analyzed for cells stably expressing LANA compared to vector control RFP cell lines from HeLa cell lines and Rat1 cell lines. The results of our studies showed that the LANA-expressing cells proliferated more rapidly in both the HeLa and Rat1 cell lines than in their corresponding vector controls (Fig. 7A and B). The rate was more dramatic in the Rat1 cell lines than in the HeLa cell line, as a reflection of the potential contribution by the human papilloma virus (HPV) antigens present in these cells. However, it is clear that the LANA-expressing cells proliferated more rapidly in both the HeLa and Rat1 cell lines.
To determine possible changes in cell cycle profile due to LANA, the cellular DNA content was measured by propidium iodide staining and flow cytometry (Fig. 7C and D). The results indicted that the LANA-expressing cell lines constantly had 18 to 20% of the cells in S phase compared to approximately 5% of S-phase cells in vector control cell lines. The difference reflects a higher cell proliferation rate and supports a previous study which suggested that LANA can stimulate S-phase entry as monitored by bromodeoxyuridine incorporation (18). In addition, the number of LANA-expressing cells in G2/M phase and cells with more than 4N DNA was typically higher, 21%, than that in vector control cells, with about 15% confined (Fig. 7, compare panels C and D). In contrast, the number of cells with LANA in G1 phase was decreased from greater than 75% in the vector control to less than 50% in the LANA-expressing cells. These results indicated that the expression of LANA induced rapid cell proliferation and that this increase was likely related to increased chromosomal aberrations.
LANA expression leads to the downregulation of p53 levels. LANA has been shown to repress the transactivation activity of p53 (16, 28). In this study, p53 activity was found to be partially neutralized by LANA expression in both stably and transiently expressing cells. In the cells stably expressing LANA, p53 transcription activity was determined by the detection of luciferase activity from a luciferase reporter construct, pG13-Luc, containing a multimerized p53 consensus-binding site (5). This construct was transfected into BJAB cells with stable LANA expression. Transfection efficiency was monitored by cotransfection with the pEGFP vector, and each transfection was done in triplicate for four LANA-expressing clones (Fig. 8A). The transactivation activity of p53 was normalized to 1.0 and compared to the results for LANA-expressing cell lines. The results show that LANA expression suppressed p53 transactivation activity by approximately 50% in the four cell lines analyzed (Fig. 8A). This suggested that LANA may directly affect p53 activity, but there was the possibility that p53 levels were also depressed in these cells. Western blot analysis of lysates from these cell lines showed a definite reduction in levels of p53 protein compared to that in the vector control cell line (Fig. 8A). In p53–/– MEF, we introduced p53 as well as an increased amount of the LANA construct and pG13-Luc reporter plasmid. The transactivation activity of p53 in mock control (no p53 transfection) cells was normalized to 1.0 (Fig. 8B). The transactivation activity of p53 was determined to be over 18 times higher in cells transfected with p53 (Fig. 8B). Moreover, LANA expression suppressed p53 transactivation activity, and the decrease of p53 activity was dose responsive to the increased level of LANA input (Fig. 8B). Therefore, LANA may regulate levels of p53 transcription from the endogenous promoter or mediate the degradation of the p53 protein, which leads to reduced levels in stable LANA lines.
To directly determine whether LANA can repress p53 expression at the level of transcription or posttranscriptionally, a titration experiment was performed in which BJAB cells were cotransfected with an increased amount of a LANA-expressing construct with a steady amount of reporter pG13-luc. The results showed that an increase in amounts of LANA resulted in decreased levels of p53 transcription activities as determined by reporter assay in a dose-response fashion (Fig. 9A). The levels of endogenous p53 transcripts were also reduced proportionally to input LANA expression (Fig. 9B and C). These results also correlated nicely with the levels of p53 protein as shown by a corresponding reduction in p53 signal by Western blot analysis as the amount of LANA was increased from a heterologous system (Fig. 9D and E). Thus, these results suggest that LANA can repress p53 transactivation activities, at least in part by repressing its endogenous promoter, thus resulting in decreased p53 transcript levels.
DISCUSSION
The generation of cell lines stably expressing the KSHV-encoded LANA in this study allowed us to demonstrate for the first time that LANA can induce chromosomal instability in multiple cell types. The observation that dramatic rises in multinucleation, numbers of micronuclei, and abnormal levels of centrosomes and spindle poles as well as increased proliferation in stable LANA cell lines strongly suggests that constitutive expression of LANA during latent infection may have a predominant role in driving the oncogenic process mediated by KSHV.
Chromosomal instability, a particular hallmark of human cancer, has been considered an adaptive response of cancer cells to environmental pressure (27). Chromosomal instability has been typically defined as an accelerated rate of gains and losses of whole or large portions of chromosomes in the context of continuous cell growth (44). In most cases, chromosomal instability results from numerous chromosomal alterations, as well as additional structural alterations. Defects in chromosome segregation, DNA damage repairing, telomere stability, and cell cycle disturbance are some of the major cellular mechanisms that can contribute to the induction of chromosomal instability (22). Loss of p53 function is a frequent and important event in the genesis and progression of many human malignancies, and a number of studies have suggested that p53-mediated cell cycle control plays a critical role in the preservation of chromosomal stability. Intact p53 responses typically impose barriers to cells with chromosome alterations by stimulating G1/S or G2/M checkpoint responses, mitotic catastrophe, and apoptotic cell death (13). In contrast, cells with an inactivated p53 response may overcome these checkpoints and maintain proliferation regardless of the chromosomal aberrations (54). This is confirmed by the observation that cells lacking p53 or having altered p53 activities have an increased ability to reenter the cell cycle following prolonged spindle disruption and can therefore initiate another round of DNA replication (7, 39, 47).
In the context of gammaherpesvirus oncogenesis, chromosomal instability has been seen in KSHV-associated malignancies; this instability is most likely mediated by one or more of its latent genes. For example, v-cyclin, one of the latently expressed genes, has been shown to block cytokinesis and drive DNA replication in human and mouse primary cells, thus stimulating polyploidy (49, 50). The abnormality triggers p53-dependent apoptosis and growth arrest, and its tumorigenesis is restrained in a p53-dependent manner (49). This is further supported by the fact that the aneuploid population survived and expanded both in vitro and in vivo in the absence of p53, leading to an oncogenic phenotype (49, 50). Therefore, mechanisms for overcoming the p53-dependent checkpoints are likely to be crucial for tumor development in KSHV-associated cancers. LANA has been shown to suppress the transactivation activity of p53 and to protect against p53-mediated cell death and is resistant to p53-dependent apoptosis but not p53-independent apoptosis (16). Elucidating the effect of constitutive LANA expression on p53 transactivation activity and chromosomal integrity will be important and will contribute to understanding the oncogenic properties of KSHV.
To provide insights into LANA's effect on inactivation of p53, this study demonstrated that LANA can induce chromosomal instability by repressing the transcription of p53 from its endogenous promoter. In multiple cell types with different backgrounds where stable cell lines were generated, aneuploidy and defects in spindle poles and centrosomes were consistently increased. These aberrations were more significant in the rat fibroblast cell line Rat1 and murine embryonic fibroblasts with wild-type or null p53 than those seen in HeLa cells. HeLa is a cervical-cancer cell line with high-risk HPV infection and consistent E6 and E7 expression. While high-risk HPV E6 protein disrupts the p53 protein by promoting p53 ubiquitin-mediated degradation, E7 protein disrupts the pRb pathway (15, 46). Previous studies showed that the E6 and E7 proteins cooperate to induce aneuploidy and chromosomal instability (14). In HeLa cells expressing LANA, a further decrease in p53 levels was consistently observed, and these results were also supported with the Rat1 cell line and MEF, suggesting a major contribution by LANA to p53 levels. The increase of aneuploidy was also corroborated by DNA content analysis, which showed that in the LANA-expressing cells, the DNA content in cells with greater than 4N was almost doubled, supporting the chromosomal changes in morphology seen with nuclear staining.
Studies have shown that the amino terminus of LANA mediates chromatin binding and that the carboxyl terminus mediates binding to a 13-bp sequence in the terminal repeats of the viral episome, anchoring the viral episome to the host chromosome (4, 11). The p53-interacting region has been mapped to the C terminus of the LANA protein (6). The N-terminal domain of LANA can neither bind p53 nor antagonize the transactivation activity of p53 (16). The C-terminal domain of the derivative LANA construct used in this study has been shown to retain the ability to bind p53 and downregulate p53 transactivity (6). Our results showed that the C terminus of LANA plays a major role in the induction of chromosomal instability, suggesting that the C terminus is in large part responsible for inducing these abnormalities, which are closely related to its ability to counteract p53 transactivation.
In the LANA-positive cell lines with chromosomal instability, p53 transactivation activity was downregulated. These results are consistent with previous microarray results which showed low p53 transcript levels in LANA-expressing cells (45). p53 function has been determined to be regulated in multiple ways. Virus-encoded oncoproteins (SV40 large T antigen, adenovirus E1B, HPV E6, and hepatitis B virus X) as well as the cellular oncoprotein Mdm-2 have been known to inhibit p53 function via physical interaction, abrogating the specific DNA binding or transactivation activity of p53. This sequesters p53 in the cytoplasm or accelerates its degradation (38). In KSHV, LANA was colocalized with p53 in tissue from KS patients, PEL cells, and human KSHV-associated solid lymphoma (25, 28). In addition, physical interaction between LANA and p53 was demonstrated (6). Furthermore, the region of p53 targeted by LANA and ORF73 from herpesvirus saimiri was mapped to the domain required for tetramerization but does not affect tetramerization (6). Thus, this study suggests another mechanism for p53 regulation, in addition to a physical interaction which involves transcription downregulation.
The DNA content analysis also confirms that LANA promotes the proliferation rate by stimulating S-phase entry (52). This agrees with the results of a previous study showing that LANA can inhibit pRb function. The tumor suppressor pRb sequesters E2F, a transcription factor required for G1/S transition (41). By inhibition of pRb, LANA promotes S-phase entry and prolongs the life span of human primary endothelial cells. The inhibition of both pRb and p53 enables LANA-expressing cells to circumvent the G1/S checkpoint in addition to the apoptotic pathway, thus contributing to oncogenesis. In endothelial cells, LANA expression can be detected very early after infection (29, 32). It is tempting to speculate that LANA inhibits pRb and helps to create a cellular environment supportive of DNA replication and susceptible to the establishment of viral latency. Such a high DNA replication demand, as well as spontaneous errors in host genome replication, is likely to drive an increase in errors during chromosome segregation. Supportively, LANA then suppresses p53 function, thus allowing the cells with mitotic aberrations to survive, resulting in the accumulation of chromosomal instability.
This study also may provide clues as to why LANA is incapable of transforming endothelial cells. Several lines of evidence indicate that the inactivation of p53 function alone is not sufficient to cause chromosomal instability. Loss of p53 does not necessarily lead to aneuploidy, as targeted disruption of p53 in either diploid tumor cells or normal human fibroblasts is not typically associated with aneuploidy even after several population doublings (8). Moreover, several cancer cell lines with p53 mutations are diploid and chromosomally stable (35). Mice with heterozygous p53 burdened with a mutant form of SV40 large T antigen that retained the ability to inactivate pRb but not p53 developed tumors and showed a diploid karyotype (37). This is also supported by results which showed that in vitro-generated neoplastic cells expressing human TERT, H-ras, and SV40 oncogenes are hyperproliferative but genomically stable despite inactivation of p53 by the SV40 large T antigen. Finally, a recent study showed that cells with partial impairment of p53 function retained normal centrosome numbers as wild-type cells (35). However, when the cells were exposed to DNA damage, they developed centrosome amplification, whereas cells carrying wild-type p53 maintained normal centrosome numbers (35). Hence, p53 inactivation may not represent the only contributor to chromosomal instability but may rather facilitate chromosomal instability in cooperation with additional cellular insults. Notably, our study showed that the transactivation activity of p53 is only partially suppressed. Additional steps may be involved in the development of chromosomal instability. Chromosome aberration may initially be stimulated by cellular insults, and since the cell lines used in this study are established cell lines, it is tempting to speculate that these insults may occur more frequently than in primary cells. In addition, replication errors may also be driven by the increased replication rate induced by LANA. These events are then followed by LANA suppression of p53 transcription activity, allowing for the accumulation of aberrations and the facilitation of chromosomal instability in these cells.
In the context of KSHV pathology, KSHV infection, establishment of latency, and the expression of latent genes, such as v-cyclin and v-FLIP, LANA proteins are important in driving oncogenesis (Fig. 10). The expression of these latent genes drives cells to undergo rapid proliferation, replication, and abortive cytokinesis and adapt from the defect in cytokinesis. Accompanied by other spontaneous cellular errors, these aberrations can trigger the activation of p53 followed by concurrent growth arrest and apoptosis. However, suppression of p53 transcript levels by LANA expression leads to a failure to monitor the generated aberrations by p53-dependent checkpoints, thus resulting in the formation of a polyploid/aneuploid population of cells with amplified numbers of centrosomes. The accumulated abnormalities lead to chromosomal instability and KSHV-associated cancers.
ACKNOWLEDGMENTS
This work was supported by grants from the Leukemia and Lymphoma Society of America and by public health service grants NCI CA072510 and CA091792 and NIDCR DE01436 (to E.S.R.). E.S.R. is a scholar of the Leukemia and Lymphoma Society of America.
We also thank Jianping Wang for her technical assistance and other members of the Robertson lab, especially Subash Verma, for helpful suggestions.
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