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Mol Cell Biol. Oct 2006; 26(20): 7506–7519.
Published online Aug 7, 2006. doi:  10.1128/MCB.00430-06
PMCID: PMC1636856

Apoptosis Inhibition by the Human DEK Oncoprotein Involves Interference with p53 Functions[down-pointing small open triangle]


The DEK proto-oncogene has been associated with human carcinogenesis—either as a fusion with the CAN nucleoporin protein or when transcriptionally upregulated. Mechanisms of intracellular DEK functions, however, have remained relatively unexplored. We have recently demonstrated that DEK expression is induced by the high-risk human papillomavirus (HPV) E7 protein in a manner which is dependent upon retinoblastoma protein function and have implicated DEK in the inhibition of cellular senescence. Additionally, overexpression of DEK resulted in significant life span extension of primary human keratinocytes. In order to determine whether DEK expression is required for cellular proliferation and/or survival, we monitored cellular responses to the knockdown of DEK in cancer and primary cells. The results indicate that DEK expression protects both HPV-positive cancer and primary human cells from apoptotic cell death. Cell death in response to DEK depletion was accompanied by increased protein stability and transcriptional activity of the p53 tumor suppressor and consequent upregulation of known p53 target genes such as p21CIP and Bax. Consistent with a possible role for p53 in DEK-mediated cell death inhibition, the p53-negative human osteosarcoma cell line SAOS-2 was resistant to the knockdown of DEK. Finally, expression of a dominant negative p53 miniprotein inhibited DEK RNA interference-induced p53 transcriptional induction, as well as cell death, thus directly implicating p53 activation in the observed apoptotic phenotype. These findings suggest a novel role for DEK in cellular survival, involving the destabilization of p53 in a manner which is likely to contribute to human carcinogenesis.

The human DEK proto-oncogene was originally identified as a fusion with the CAN nucleoporin protein in a subset of acute myeloid leukemia patients carrying the t(6;9) translocation (50). Since its discovery, DEK has also been found to be transcriptionally upregulated in a number of aggressive human tumors such as bladder carcinoma, hepatocellular carcinoma, glioblastoma, melanoma, and acute myeloid leukemia types that do not exhibit the above translocation (21, 29, 31, 32, 44). DEK is a potential target of chromosome 6p22 gains in retinoblastoma, as well as in bladder cancer, suggesting that DEK gain of function may provide a selective advantage for tumor development (15, 20, 41). Consistent with potential oncogenic DEK properties is the developmental regulation of DEK mRNA with high expression in immature retinal mouse cells and with induced levels of DEK expression in simian virus 40 (SV40) large T antigen-induced retinoblastomas (41). The latter finding may further support our previous results obtained with primary human keratinocytes, where upregulated expression of DEK in response to the SV40 T antigen-related high-risk human papillomavirus (HPV) E7 protein was dependent upon retinoblastoma protein function (59). Furthermore, our data indicated that both the replicative senescence of primary human foreskin keratinocytes (HFKs) and senescence of HeLa cervical cancer cells are accompanied by DEK repression. DEK overexpression partially inhibited HeLa cell senescence and extended the life span of primary human keratinocytes, implicating this molecule functionally as a senescence inhibitor.

The wild-type 43-kDa DEK phosphoprotein is depicted schematically in Fig. Fig.1A.1A. Most intracellular DEK protein is associated with chromatin, whereas about 10% is associated with RNA (26). DEK can interact with any DNA template but exhibits a particularly high affinity for cruciform and supercoiled DNA (52). DEK binding causes the introduction of positive DNA supercoils, stimulates the ligation of linear DNA molecules, and converts DNA circles into catenated DNA in the presence of topoisomerase I (52, 53). Two independent DNA binding domains exist within the protein, and the ability of DEK to interact with DNA is reduced in vitro by phosphorylation, as well as acetylation (9, 27, 28). In vivo, DEK phosphorylation peaks in the G1 phase of the cell cycle but without any significant overall alteration of DEK interactions with chromatin (27). According to a model that incorporates a combination of DEK studies, reduced DNA binding by phosphorylated DEK in G1 may be masked by a compensating event whereby DEK phosphorylation favors DEK multimerization, which in turn stimulates the tethering of DEK to chromatin (54). It will be interesting to define specific roles for DEK phosphorylation with respect to its normal and cancer-related cellular activities. Intracellular DEK acetylation leads to DEK relocation into interchromatin granules, nuclear substructures with presumptive roles in transcription and mRNA processing (9). Taken together, these findings suggest that posttranscriptional modification of the human DEK proto-oncogene may play important roles in transcription, mRNA processing, and chromatin modulation, all of which have been suggested to be regulated and possibly even coordinated by DEK (2, 6, 23, 36, 45a, 53, 54).

FIG. 1.
DEK depletion represses HeLa cell growth. (A) Schematic representation of the human DEK protein. The conserved SAP DNA binding domain is indicated. A separate C-terminal DNA binding domain has been mapped to aa 270 to 350 (54). Several highly acidic domains ...

The human p53 protein is the most frequently inactivated tumor suppressor gene in human cancer (4, 25) and is involved in the control of cellular proliferation in response to stress (for reviews, see references 40 and 51). During carcinogenesis, p53 can be inactivated through various mechanisms. These include (i) mutations in the p53 DNA binding domain, (ii) sustained inactivation by viral oncogenes such as HPV E6, (iii) inhibition of p53 activation through deletion of p14ARF, and (iv) mutational inactivation of critical downstream effectors such as BAX (for reviews, see references 34, 48, and 49). In response to intra- or extracellular stressors such as DNA damage, aberrant oncogene expression, telomere erosion, or loss of survival factors, p53 protein is activated through upregulated protein levels and/or posttranslational modifications. One important response to p53 activation is cellular apoptosis, or programmed cell death, while others include cell cycle arrest, senescence, and differentiation. How these various programs are selectively induced is still under investigation, but the final decision is at least in part dependent upon the cell type affected and the nature of participating apoptotic signaling pathways. p53 can exert its functions via promoter binding, causing the direct transcriptional induction of specific target genes such as MDM2, p21CIP1, and proapoptotic Bcl-2 family members. The role of p53 in apoptosis induction also extends to nontranscriptional mechanisms, where p53 translocates to the mitochondrial membrane and interacts with the proapoptotic factors Bax and Bid (for a review, see reference 37). This results in permeabilization of the mitochondrial membrane, release of cytochrome c, and activation of effector caspases, ultimately leading to apoptosis execution.

We have previously shown that DEK message and protein levels are significantly reduced during HPV-positive HeLa cell, as well as primary cell, senescence. DEK overexpression resulted in the partial inhibition of HeLa cell senescence and extended the life span of primary human keratinocytes, thus implicating DEK as a senescence inhibitor. In order to determine whether DEK expression is required for cancer cell growth, we employed RNA interference (RNAi) approaches for specific targeting of DEK in cancer and primary cells. DEK depletion in HeLa cervical cancer cells resulted in the induction of apoptosis. Similar results were obtained with primary human keratinocytes, implicating DEK as a cancer and primary cell survival factor. The observed apoptotic phenotype was accompanied by p53 stabilization and elevated transcriptional activity and was only minimally induced in p53-negative SAOS-2 cells. Primary keratinocyte apoptosis in response to DEK depletion was inhibited via expression of a dominant negative p53 protein, thus implicating p53 as a DEK target. Taken together, our results uncover a new role for DEK in the inhibition of apoptosis via destabilization of p53. Our data imply that potential anticancer treatments via targeting of DEK may depend upon the status of p53 in the respective cancers and may be accompanied by significant toxicity.


Cell culture.

The human HeLa cervical cancer cell line was maintained in Dulbecco's modified Eagle medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) and antibiotics. The human osteosarcoma cell line SAOS-2 (American Type Culture Collection, Manassas, VA) was maintained in McCoy's 5a modified medium (American Type Culture Collection, Manassas, VA) supplemented with 15% FBS and antibiotics. Primary HFKs were prepared from human foreskins (IBC protocol CHMC 02-9-29X) and maintained in Epilife medium (Cascade Biologics, Portland, OR) and antibiotics.

Plasmid and viral constructs.

Double-stranded RNAi oligonucleotides were designed against the DEK open reading frame and were purchased from Dharmacon (Lafayette, CO). DEK small interfering RNA (siRNA) oligonucleotides comprised the sequence 5′-UGUCCUCAUUAAAGAAGAA-3′, and the respective scrambled siRNA sequence was 5′-CUCUAAAGACAGGUUAUAA-3′. For plasmid-based RNAi, a short hairpin construct was designed under control of the U6 promoter and terminating with a poly-T transcriptional termination signal according to published specifications (46, 60). The DEK targeting cassette was 5′-GGATAGTTCAGATGATGAACCCTCGAGGGTTCATCATCTGAACTATCC CTTTTTG-3′, where the underlined sequence is the spacer region. After sequence verification, the DEKsh expression cassette was excised from the BS/U6 vector with XbaI and KpnI and inserted into an adenovirus pAdTrack-CMV shuttle vector containing a green fluorescent (GFP) marker gene as described in reference 22. The cassette was resequenced, and recombination with the viral backbone in bacteria was followed up by screening for full-length viral clones and transfection of 293 cells for the generation of replication-deficient AdDEKsh virus. For expression of truncated DEK68-226, a cDNA comprising amino acids (aa) 68 to 226 was cloned into the pCMV-HA vector (Clontech Laboratories, Mountain View, CA) with EcoRI and KpnI restriction sites. The Adp53 virus was obtained from Vector Biolabs (Philadelphia, PA), and the Ad, AdE2ts, and AdDEK viruses were previously described (56, 59). Viral titers were determined by plaque assays on 293 cells and/or by flow cytometry 1 day postinfection with GFP detection. Correspondingly, viral titers were expressed as either PFU or infectious units (IUs). Quantitation of IUs was done by the following calculation: (% GFP-positive cells × total cell number at infection × virus dilution)/virus stock volume used. The empty LXSN virus was described previously (59), and the LXSN-p53dd virus was a generous gift from Moshe Oren (19).

Transient transfections.

Cells on 60-mm plates were transfected with a total of 4 μg of plasmid DNA or siRNA oligonucleotides with XtremeGENE (Roche Diagnostics, Indianapolis, IN) by following the manufacturer's instructions. For cotransfections, the cells were transfected with 1 μg of puromycin resistance plasmid plus 3 μg of DNA or with 500 ng of neomycin resistance plasmid plus 3.5 μg of DNA. Cells were selected with medium containing 400 ng/ml puromycin for 5 days or with medium containing 900 μg/ml G418 for 9 days, respectively.

Adenovirus and retrovirus infections.

For adenovirus infections, cells were washed with phosphate-buffered saline (PBS) and infected with an adenovirus stock containing the indicated number of IUs or PFU per cell in PBS containing 4% FBS for 1 h. Virus was then aspirated, and the cells were washed twice with PBS and overlaid with fresh medium. For retrovirus infections, the cells were infected with 1 ml of LXSN virus or with 1 ml of LXSN-p53dd virus for 4 h in medium containing 8 μg/ml Polybrene. The cells were washed with PBS and overlaid with fresh medium. One day after infection, the cells were overlaid with medium containing 900 μg/ml G418 for HeLa cells or 200 μg/ml G418 for primary keratinocytes.

Northern blot analysis.

Total RNA was isolated with Trizol reagent (BRL, Bethesda, MD) according to the manufacturer's instructions. A total of 10 μg of RNA was resolved on a 1.2% agarose gel containing formaldehyde, transferred to a Duralon charged nylon membrane from Stratagene (La Jolla, CA) and UV cross-linked. The membrane was hybridized with a randomly primed 32P-labeled cDNA probe comprising the full-length DEK open reading frame, washed, and exposed according to standard protocols. Membranes were stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control.

Western blot analysis.

The cells were washed with PBS, and whole-cell lysates were harvested with RIPA buffer (1% Triton, 1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 0.16 M NaCl, 10 mM Tris [pH 7.4], 5 mM EDTA) supplemented with protease inhibitor cocktail (Pharmingen, San Diego, CA). Protein concentrations were determined with Bradford reagent (Bio-Rad, Hercules, CA). Aliquots containing equal amounts of total protein were boiled in SDS sample buffer and resolved by SDS-polyacrylamide gel electrophoresis. Proteins were then transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) and probed overnight with either the DEK monoclonal antibody (BD Biosciences, San Diego, CA), the HA monoclonal antibody (Sigma, St. Louis, MO), the p53 (Ab-6) monoclonal antibody (Calbiochem, San Diego, CA), the p21 polyclonal antibody (Pharmingen, San Diego, CA), the BAX (6A7) monoclonal antibody (Pharmingen, San Diego, CA), the p16 monoclonal antibody (Pharmingen, San Diego, CA), or the actin-specific monoclonal antibody, a generous gift from James Lessard. On the next day, the membranes were washed with TNET (10 mM Tris, 2.5 mM EDTA, 50 mM NaCl, 0.1% Tween 20) and secondary anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase (Amersham, Piscataway, NJ) were added for 30 min. Membranes were then exposed to enhanced-chemiluminescence reagents (Perkin-Elmer, Boston, MA), and the protein bands were detected by autoradiography.

Luciferase reporter assays.

HeLa cells or HeLa cells stably transduced with the LXSN and LXSN-p53dd retroviruses were plated in six-well dishes and transfected with 2 μg of the indicated luciferase reporter plasmids, including the pGL3-Basic control vector (Promega Corporation, Madison, WI), the p21CIP1 reporter pGL2-p21luc (14), and the p53 reporter PG13-luc (13), kind gifts from Bert Vogelstein; the maspin reporter (pM-luc) (62), a generous gift from Shiv Srivastava; the HDM2-luc reporter (61), a generous gift from Moshe Oren; and the PTGFβ reporter constructs (47), either wild type or with the p53 binding site deleted, generous gifts of Yi Sun. The PTGFβ wild-type and PTGFβp53−/− mutant constructs were originally called PTGFβ W/p53BS and PTGFβ W/Op53BS. On the following day, the cells were infected with empty Ad, AdE2ts, AdDEK, or AdDEKsh adenovirus. The cells were harvested 3 days postinfection, counted, and resuspended in cell lysis buffer containing 25 mM Tris-PO4 (pH 7.8), 15% glycerol, 2% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 1% lecithin, 1% bovine serum albumin, 4 mM EGTA, 8 mM MgCl2, 1 mM dithiothreitol, and 0.4 mM phenylmethylsulfonyl fluoride. The amount of lysis buffer was normalized to the cell number, with 200 μl for the Ad control. Cell lysates were incubated at room temperature for 15 min, freeze-thawed three times, and cleared of cell debris by spinning at 4°C for 5 min. For the luciferase assays, 25 μl of cell lysate was added to 175 μl of luciferase assay buffer (25 mM Tris/phosphate, 20 mM MgSO4, 4 mM EGTA, 2 mM ATP, 1 mM dithiothreitol) in 96-well Greiner Lumintrac 200 plates (Greiner, Longwood, FL). Analysis was performed with a Luminoskan luminometer (Lab Systems, Franklin, MA).

Cell cycle analysis by flow cytometry.

Cells were infected with either empty Ad, AdDEK, or AdDEKsh virus. After 3 to 7 days, depending upon the experiment, the cells were detached with PBS-0.1% EDTA at 37°C, washed with PBS, counted on a hemocytometer, and fixed overnight in cold 80% ethanol at 4°C. The cells were then pelleted by centrifugation, washed twice with PBS-1% BSA, resuspended in 800 μl of PBS-1% BSA-100 μl of propidium iodide stock solution (500 μg/ml propidium iodide in 10 mM sodium citrate, pH 7.0)-100 μl of boiled RNase A (10 mg/ml prepared in 10 mM Tris-HCl, pH 7.5) at a concentration of 106 cells/ml, and incubated at 37°C for 30 min. Analysis was performed with BD Cell-Quest software on a Flow Cytometer (BD Biosciences, San Jose, CA).

Apoptosis analysis by flow cytometry.

Cells were either transfected with empty BS/U6, BS/U6-GFPsh, or BS/U6-DEKsh plasmid or infected with either empty Ad, AdDEK, or AdDEKsh. Transfected or infected cells were harvested by trypsinization on the indicated days, washed in cold PBS, counted on a hemocytometer, and fixed with BD Cytofix/Cytoperm (BD Pharmingen, San Diego, CA) for 20 min at room temperature. The cells were then washed with BD Perm/Wash buffer twice and incubated with 20 μl/106 cells of either fluorescein isothiocyanate-conjugated or biotinylated anti-active Caspase 3 antibody (BD Pharmingen, San Diego, CA) for 1 h at room temperature in the dark. Where biotinylated antibody was used, the cells were washed and incubated with 5 ng/106 cells of streptavidin-allophycocyanin (APC; BD Pharmingen, San Diego, CA) for 30 min at room temperature. As a control for nonspecific antibody binding, a sample was stained with the streptavidin-APC without anti-active caspase 3 primary antibody addition. Alternatively, 106 cells were fixed as described above and 0.25 μg of 7-aminoactinomycin D (7-AAD) in a volume of 20 μl was added (BD Pharmingen, San Diego, CA). The sub-G1 population was gated for quantitation of the percentage of apoptotic cells. Since 100% of the cell population was infected, gating for GFP expression was not performed. The cells were analyzed with BD Cell-Quest software on a Flow Cytometer (BD Biosciences, San Jose, CA).

MTS assays.

The CellTiter 96 Aqueous One assay (Promega, Madison, WI) was used for assessment of cellular viability in response to DEK depletion. The assay is based on addition of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), and the electron coupling reagent phenazine methosulfate and measures dehydrogenase enzyme activity found in metabolically active cells. MTS is chemically reduced by cells into formazan, which is soluble in tissue culture medium. Measurement of the absorbance of the formazan, proportional to the number of living cells, can be carried out in 96-well microplates at 490 nm. The cells were infected as indicated, and 2,000 cells/well were plated in a 96-well dish. MTS assays were performed on the indicated days, and the plates were incubated at 37°C in a humidified 5% CO2 atmosphere. Absorbance at 490 nm was recorded 3 h later on an enzyme-linked immunosorbent assay plate reader (Molecular Dynamics, Menlo Park, CA).

Transient cellular growth assays.

HeLa and HFK cells plated in 12-well dishes were infected with either empty Ad, AdDEK, or AdDEKsh. After trypan blue staining, viable cells were counted on 7 consecutive days.

Colony reduction assays.

HeLa cells were infected as described above for cell cycle analyses or transfected with empty vector or BS/U6-DEKsh alone or in combination with CMV-HA.DEK68-226 along with 500 ng of neomycin selection plasmid. Infected cells were split 1:50 24 h later. Transfected cells were split 1:6 24 h later and selected in G418-containing medium for 3 weeks. Colonies were stained with methylene blue and counted.

Kinetic analysis of p53 stability.

HeLa cells were infected with empty Ad or AdDEKsh virus and then exposed to 50 μg/ml cycloheximide at 4 days postinfection. At the indicated time points, whole-cell protein extracts were harvested and subjected to Western blot analyses for detection of p53 and actin. The resulting signals were quantitated with NIH Image and are presented as fold changes over the amount of protein at time zero.


DEK depletion results in inhibition of HeLa cell proliferation.

Our previous experiments have implicated DEK as an inhibitor of cellular senescence whose overexpression resulted in extension of the life span of primary human keratinocytes (59). Unlike primary cells, HeLa and various other cancer cell lines (data not shown) exhibit high levels of DEK expression. We utilized an RNAi approach to determine whether HeLa cell growth requires DEK expression. Cells were transfected with DEK-specific double-stranded siRNA, a corresponding scrambled DEK siRNA control, or a nonspecific siRNA. Control transfections with fluorescently labeled oligonucleotides indicated transfection efficiencies of >95%. Successful knockdown of intracellular DEK protein levels was confirmed by Western blot analysis (Fig. (Fig.1B).1B). DEK protein levels were indeed repressed in response to DEK siRNA compared to cells that were transfected with either of the control siRNAs (compare lane 3 with lanes 1 and 2). Cytotoxicity assays were performed at various time points following transfection and revealed complete inhibition of cellular proliferation and/or survival in response to DEK depletion (Fig. (Fig.1C1C).

Apoptosis is a consequence of DEK depletion.

In order to extend our RNAi approach to other cell lines and to employ coselection methods, we constructed a DEK-specific short hairpin plasmid according to published specifications (46, 60). We used Northern blot analysis to verify successful DEK knockdown following the transfection of HeLa cells. As shown in Fig. Fig.2A,2A, DEK message levels were reduced in the presence of the BS/U6-DEKsh plasmid compared to the empty-vector control (Fig. (Fig.2A,2A, compare lanes 1 and 2 for DEK mRNA, as well as for the GAPDH loading control). In order to assess effects of DEK mRNA reduction on DEK protein levels and to determine the timing of DEK depletion, we cotransfected HeLa cells with the BS/U6-DEKsh plasmid along with a puromycin resistance plasmid, followed by selection for a total of 5 days. Protein lysates were harvested on each day, and equal amounts of protein were subjected to Western blot analysis with a DEK-specific monoclonal antibody (Fig. (Fig.2B).2B). Identical results were obtained with a single band at around 45 kDa upon probing with a DEK-specific polyclonal antibody (data not shown). A decrease in DEK protein levels was evident on day 3 posttransfection of DEKsh but not the control vectors, and DEK levels continued to decline over the course of the experiment. Significant loss of viable cells occurred as a result of DEK depletion in these experiments (data not shown). Since we had previously associated DEK repression with HeLa cell senescence, we determined whether apoptosis occurred in response to DEK RNAi (Fig. (Fig.2C).2C). HeLa cells were transfected with backbone plasmid BS/U6, a BS/U6-GFPsh-specific control plasmid, or plasmid BS/U6-DEKsh in the absence of a selectable marker plasmid, and caspase 3 activation was quantitated on each day for a total of 5 days posttransfection. No antibiotic selection was applied to this experiment, and the transfection efficiency was greater than 90%. Whereas the GFPsh control plasmid induced apoptosis in a minor fraction of the total cell population over time, almost 45% of the cells underwent apoptosis in the presence of plasmid DEKsh. In order to confirm that the apoptosis phenotype was indeed due to DEK depletion and was not a result of the nonspecific targeting of other molecules, we overexpressed a truncated DEK protein comprising aa 68 to 226 in an attempt to overcome the cell death phenotype (Fig. (Fig.2D).2D). This protein is fully functional for introduction of positive supercoils into DNA templates in vitro (10, 28), but its mRNA was not expected to be susceptible to the above RNAi approach because of the absence of the targeted sequence. HeLa cells were cotransfected with either empty vector BS/U6, the vector DEKsh, or the vector DEKsh plus the truncated DEK68-226 vector along with a neomycin resistance plasmid. The cells were split and selected in G418-containing medium. After 2 weeks, a dramatic colony reduction was observed in the presence of the DEKsh vector compared to the control, which was partially overcome in the presence of the DEK68-226 overexpression plasmid. Taken together, these data demonstrated that DEK knockdown in HeLa cells results in apoptosis induction and thus implicate DEK as a cellular apoptosis inhibitor.

FIG. 2.
Cellular growth arrest following DEK depletion is due to apoptosis. (A) Northern blot analyses. HeLa cells were transfected with either an empty vector (lane 1) or the DEKsh vector (lane 2), and RNA was harvested after 2 days. Equal amounts of RNA were ...

DEK depletion does not result in cell cycle aberrations.

In order to eliminate background problems that are associated with transfections and to ultimately extend these experiments to nontransfectable primary cells, we generated an adenovirus construct containing the DEKsh/U6 cassette. We first verified AdDEKsh vector functionality by infecting HeLa cells with increasing multiplicities of infection (MOIs) ranging from 3 to 30 PFU with both empty adenovirus and the AdDEKsh virus. DEK protein levels were reduced in the AdDEKsh-infected cells compared to those in empty-Ad-infected cells at all MOIs (Fig. (Fig.3A).3A). We next compared short-term HeLa cell growth in response to Ad versus AdDEKsh infection (Fig. (Fig.3B).3B). Starting on day 3, there was a significant decrease in cellular growth in the DEK-depleted cells compared to the control cells, in correlation with our RNAi data obtained with transfected siRNAs or the DEKsh plasmid. Cells were then infected and harvested for cell cycle analyses by flow cytometry (Fig. (Fig.3C).3C). A comparison between empty-Ad- and AdDEKsh-infected cells revealed that cell cycle distributions in various phases of the cell cycle did not exhibit significant differences. In order to directly confirm apoptosis in response to DEK depletion in this system, the cells were infected and harvested for quantitation of the percentage of apoptotic cells by measurement of 7-AAD incorporation (Fig. (Fig.3D).3D). We observed significant apoptosis induction on days 3 and 4 postinfection in response to DEKsh infection compared to the controls. Taken together, these experiments indicated that cellular growth arrest following DEK depletion is due to apoptotic cell death induction.

FIG. 3.
DEK depletion does not result in aberrant cell cycle progression. (A) Western blot analyses. HeLa cells were infected with either empty Ad or AdDEKsh at an MOI of 3, 10, or 30 PFU. Total protein was harvested after 4 days and subjected to Western blot ...

DEK overexpression inhibits apoptosis in HeLa cells.

In HPV-positive cancer cells, the p53 and pRB tumor suppressors are normally retained in their wild-type configuration but are expressed at low levels because of specific E6 and E7 activities. Tumor suppressor pathways can therefore be reactivated in response to viral oncogene repression. Previous results demonstrated that DEK overexpression inhibits senescence in a HeLa cell system in which the HPV E6 and E7 oncogenes were specifically repressed (59). In order to determine whether DEK overexpression may have effects on proliferating, nonsenescent HeLa cells, we performed infections with the empty Ad or AdDEK virus, followed by cell plating at low densities. Viable-cell counts were determined for 7 consecutive days (Fig. (Fig.4A).4A). Over time, increased numbers of viable cells were observed in AdDEK-infected HeLa cells over control infected HeLa cells. The increased cell counts following DEK overexpression were reflected in increased colony numbers when sparsely seeded HeLa cells were allowed to proliferate for 3 weeks (Fig. (Fig.4B).4B). As shown previously, the detection of increased DEK protein expression after AdDEK infection is slow but detectable on day 7 postinfection (Fig. (Fig.4B)4B) (59). Cell cycle profiles were generated in order to correlate the observed DEK-mediated stimulation of cell growth with either proliferative or apoptotic effects. No differences in cell cycle distribution were observed during the relevant time frame by propidium iodide staining (Fig. (Fig.4C).4C). We quantitated apoptosis induction with an activated caspase 3 antibody and flow cytometry (Fig. (Fig.4D).4D). Between 6 and 14% apoptotic cells were detected between days 5 and 8 following empty-adenovirus infection and sparse plating. DEK overexpression resulted in a reduction of apoptosis to approximately 50% of the Ad-infected baseline levels at all times. On days 7 and 8 postinfection, when DEK overexpression is clearly detectable by Western blot analysis (Fig. (Fig.4B),4B), the observed apoptosis repression was statistically significant. These results suggested that DEK stimulates the growth of cervical cancer cells through the suppression of apoptosis and that DEK RNAi may thus be a useful approach for the selective killing of cervical cancer cells.

FIG. 4.
DEK overexpression inhibits HeLa cell apoptosis. (A) Transient cell growth assay. HeLa cells were infected with an MOI of 100 IUs of the indicated viruses and counted on each day for a total of 7 days following trypan blue exclusion. (B) Colony assays ...

SAOS-2 osteosarcoma cells are partially resistant to DEK depletion.

In order to determine whether dependence upon high levels of DEK expression is a shared property of cancer cells, we chose to analyze the effects of DEK modulation in SAOS-2 osteosarcoma cells. Levels of DEK protein expression are similar in these two cell lines (data not shown). In contrast to HeLa cells, which express p53 and pRb in their wild-type configuration but at low levels under control of the viral E6/E7 oncogenes, SAOS-2 cells carry pRb and p53 deletions. SAOS-2 cells were infected with either empty Ad, AdDEK, or AdDEKsh virus, and DEK protein levels at 4 days postinfection are shown in Fig. Fig.5A.5A. At this time point, DEK overexpression following AdDEK infection in both HeLa and SAOS-2 cells is slight, whereas DEK knockdown following AdDEKsh infection is dramatic (compare with Fig. Fig.3A3A and and4B).4B). We then compared the effects of DEK overexpression and knockdown on SAOS-2 and HeLa cell growth in both cell lines by cytotoxicity assays (Fig. (Fig.5B),5B), with the results normalized to empty-Ad-infected cells. For the AdDEK HeLa cell infections, cellular metabolic activity was increased by about 3-fold on day 3 and further stimulated to about 13-fold by day 7. This effect corresponded to low DEK expression levels early after infection, whereas significant expression was observed on day 7 at later time points (59). In contrast, stimulation of cellular growth following AdDEK expression was minor in SAOS-2 cells. Similarly, DEK knockdown in HeLa cells resulted in 21- and 33-fold decreases in metabolic activity on days 5 and 7, respectively, compared to a modest 2-fold decrease in SAOS-2 cells. Direct measurements of caspase 3 activation in similar experiments revealed significant apoptosis induction in HeLa cells due to DEK depletion, compared to a minor effect in SAOS-2 cells (Fig. (Fig.5C).5C). These experiments suggested that not all cancer cells are equally dependent upon DEK expression for survival.

FIG. 5.
SAOS-2 cells are resistant to cell death in response to DEK depletion. (A) Western blot analyses. SAOS-2 cells were infected with either empty Ad, AdDEK, or AdDEKsh (lanes 1 to 3) at an MOI of 50 IUs. Total protein extracts were harvested after 4 days ...

Survival of primary human keratinocytes is dependent upon DEK expression.

We had previously observed that DEK expression levels are low in primary human keratinocytes and fibroblasts, as well as in primary mouse embryo fibroblasts (MEFs), compared to cervical cancer cells (data not shown) (59). Additionally, overexpression of DEK in HFKs resulted in a significant life span extension. In order to assess whether the targeting of DEK might be of therapeutic interest and associated with low toxicity in primary cells, we measured the effects of DEK overexpression and knockdown in primary HFKs. The cells were infected with empty Ad, AdDEK, or AdDEKsh, and protein lysates harvested on day 4 postinfection were subjected to Western blot analysis (Fig. (Fig.6A).6A). DEK overexpression and knockdown were observed with the respective vectors, similar to previous results obtained with HeLa and SAOS-2 cells (compare with Fig. Fig.3A,3A, ,4B,4B, and and5A).5A). Short-term cellular growth assays by cell counting revealed a stimulation of HFK growth upon DEK overexpression and substantial loss of cell growth upon DEK knockdown (Fig. (Fig.6B).6B). Measurements of caspase 3 activation implicated apoptosis in the loss of cellular growth in response to DEK depletion on day 5 (Fig. (Fig.6C)6C) and protection from apoptosis upon DEK overexpression on day 7 (Fig. (Fig.6D),6D), thus arguing that a role for DEK in cell survival is not unique to cancer cells.

FIG. 6.
DEK depletion results in apoptosis in primary human keratinocytes. (A) Western blot analyses. HFKs were infected at an MOI of 100 IUs with the indicated viruses and subjected to DEK-specific Western blot analysis on day 4 postinfection. (B) Transient ...

DEK inhibits p53 transcriptional activity.

Given the known proapoptotic role of the human p53 tumor suppressor and our observation that DEK effects were minor in p53-negative SAOS-2 cells, we investigated whether DEK expression could modulate p53 transcriptional activity (Fig. (Fig.7A).7A). A panel of p53-responsive reporter plasmids containing either an artificial p53-driven promoter (PG13) or promoter regions derived from the endogenous p53-responsive human MDM2, maspin, p21CIP1, and PTGFβ promoters were used for these experiments. The cells were transfected with various reporter constructs and superinfected with empty Ad, AdDEK, or AdDEKsh, and relative luciferase activities were then quantitated as described in Materials and Methods. DEK modulation did not significantly affect the relative transcriptional activities of the backbone vector pGL3. However, transfection of the p53-responsive reporters revealed transcriptional repression in response to DEK overexpression. Conversely, significant stimulation of p53 transcriptional activities was observed in response to DEK knockdown. Even though the observed trends were consistent among the p53 reporter genes, the degree of transcriptional stimulation or repression varied, indicating that DEK modulation may affect different p53 target genes to various extents. Importantly, dramatic effects on the known p53-inducible PTGFβ promoter were observed not with the corresponding PTGFβp53−/− reporter containing the same promoter but with a deletion of two relevant p53 binding sites at nucleotides −890 and −30 relative to the transcriptional start site (47). These results implicated DEK in the regulation of p53 transcriptional activity. In order to further examine effects of DEK modulation on p53 transcription in these experiments, we made use of a retrovirus construct encoding a dominant negative truncated form of p53 (19). This p53dd miniprotein specifically interferes with the sequence-specific promoter binding of p53 in mouse and human cells. HeLa cells were stably transduced with either empty LXSN retrovirus or LXSN-p53dd and selected in G418-containing medium. We first verified LXSN-p53dd retrovirus functionality in the selected cell population by artificially elevating HeLa cell p53 protein levels. For this purpose, we used a temperature-sensitive E2 protein that is known to repress E6/E7 expression and consequently activate p53 at the permissive temperature (56) and assessed the degree of p53dd interference in this system (Fig. (Fig.7B).7B). The p53-responsive PTGFβ reporter plasmid was used for these experiments. Following reporter gene transfection, the cells were superinfected with either Ad or AdE2ts. Consistent with the reported E2 effects on p53 activation, a threefold upregulation of PTGFβ reporter activity was indeed observed following AdE2ts infection and was inhibited in the p53dd-transduced cells, thus confirming p53dd functionality (Fig. (Fig.7B).7B). In order to assess effects of p53dd expression on DEKsh-mediated promoter activation, control and p53dd-transduced HeLa cells were again transfected with the PTGFβ reporter and superinfected with Ad versus AdDEKsh (Fig. (Fig.7C).7C). PTGFβ promoter gene activation in response to DEK RNAi was significantly repressed by the dominant negative p53 protein, ruling out direct effects of DEK on individual promoter constructs. Taken together, these results suggested that the human DEK proto-oncogene acts as a repressor of p53 transcriptional activities.

FIG. 7.
DEK inhibits p53 transcriptional activity and the expression of several p53 target genes. Luciferase (luc.) reporter assays. (A) HeLa cells were transfected with the indicated p53-responsive promoter luciferase constructs as described in Materials and ...

Effects of DEK modulation on endogenous p53 target genes.

In order to determine whether the observed DEK-mediated regulation of p53-responsive reporter genes is relevant, we next performed Western blot analyses for the detection of endogenous p53 target genes (Fig. (Fig.7D).7D). Low but detectable levels of wild-type p53 are present in HeLa cells because of p53 degradation by the HPV type 18 E6 oncoprotein. p53 protein levels became undetectable upon DEK overexpression and were significantly increased in response to DEK depletion, suggesting that DEK negatively regulates intracellular levels of p53. Increased p53 expression in response to DEK depletion was accompanied by increased expression of its endogenous target p21CIP1 but not of the p16INK4A cyclin/Cdk kinase, which is not regulated by p53. As expected, similar responses were not observed in p53-negative SAOS-2 cells. Since reduced p53 stability in HeLa cells is governed by the presence of HPV E6, it was possible that DEK effects on E6 might indirectly be responsible for the observed p53 regulation. We therefore examined the consequences of DEK overexpression and depletion on p53 protein levels in primary HFKs (Fig. (Fig.7E).7E). DEK overexpression reduced p53 protein levels, and conversely, DEK depletion upregulated p53 protein levels with a corresponding upregulation of its downstream targets p21CIP1 and BAX. These findings suggest that the human DEK oncogene negatively regulates p53 protein expression via a mechanism that is shared by cancer and primary cells.

DEK regulates p53 at the level of protein stability.

In order to study the mechanism of p53 inhibition by DEK in more detail, we determined DEK effects on exogenously overexpressed p53 protein with adenovirus expression vectors. Primary HFKs were infected with AdDEK and/or Adp53, and levels of DEK and p53 protein expression were monitored over time. Both p53 and DEK were overexpressed at all time points in response to infection with the relevant vectors. Whereas p53 levels were only slightly affected by DEK on day 5, we observed a clear reduction in p53 protein levels on subsequent days, suggesting that DEK-mediated p53 regulation occurs at a posttranscriptional level. In order to test this hypothesis directly, we measured the half-life of p53 in HeLa cells under normal versus DEK-depleted conditions. The cells were infected with either empty Ad or AdDEKsh and exposed to the protein synthesis inhibitor cycloheximide. Protein lysates were taken at several time points, and p53 protein levels were quantitated. Whereas p53 protein levels declined to 20% of the original amount in control infected cells, DEK depletion resulted in a very modest reduction to only 85% after 120 min. These data suggested that DEK-mediated destabilization of p53 protein underlies the observed transcriptional inhibition of p53 target genes in Fig. Fig.77.

Apoptosis in response to DEK depletion involves p53.

Results from Fig. Fig.55 to to88 had correlated p53 upregulation with apoptotic cell death. In order to demonstrate directly a functional role for p53 in the observed phenotype, we investigated whether expression of the dominant negative p53 protein in primary keratinocytes could inhibit apoptosis in response to DEK depletion. Cell pools that were transduced either with the empty vector or with the LXSN-p53dd vector were infected with Ad or AdDEKsh. Apoptosis was quantitated on day 5 post adenovirus infection by flow cytometry for the detection of activated caspase as a readout (Fig. (Fig.9A).9A). Pictures of infected cells were taken at the same time point and are shown in Fig. Fig.9B.9B. Expression of dominant negative p53 clearly inhibited the apoptosis phenotype in a manner which was more dramatically observed phenotypically in Fig. Fig.9B9B compared to the measurements of caspase 3 activation in Fig. Fig.9A.9A. This difference likely represents the outgrowth of a surviving cell population at this relatively late time point. Our data support a role for DEK as a negative regulator of p53 activities in a manner that influences cellular survival.

FIG. 8.
DEK modulation affects p53 protein stability. (A) Western blot analyses. Primary HFKs were infected with empty Ad, AdDEK, and Adp53 expression vectors, either alone or in combination, and harvested on the indicated days. Protein lysates were subjected ...
FIG. 9.
Apoptosis in response to DEK depletion requires p53. (A) Apoptosis assay. Primary HFKs were stably transduced with either LXSN or dominant negative p53dd retrovirus and then superinfected with either empty Ad or AdDEKsh. Apoptosis was quantitated with ...


A number of human diseases have been associated with DEK modulation, including several cancers exhibiting upregulated DEK expression or the DEK-CAN fusion, as well as human autoimmune diseases with a striking presence of DEK-specific autoantibodies (3, 11, 12, 21, 29, 31, 32, 44, 58). Recent reports have implicated DEK in the episomal maintenance of Kaposi's sarcoma herpesvirus as a tether between the latency-associated nuclear antigen and chromosomes (30) and in specific binding to the human immunodeficiency virus type 2-inducible enhancer, which may relate to viral gene regulation (9, 16, 17). Functions of the human DEK proto-oncogene have been well characterized in vitro and have indicated important roles in the regulation of chromatin architecture. DEK induces positive supercoils in DNA templates in a manner that does not require ATP but requires the activity of a eukaryotic type I topoisomerase. It is likely that this is the activity which is responsible for the inhibition of SV40 DNA replication in vitro (2, 53). In addition to potential roles in DNA replication, DEK has been implicated as a transcriptional regulator that is enriched on transcriptionally active chromatin (26) and present within specific transcription factor complexes. DEK has been described both as a transcriptional stimulator (6, 24) and as a transcriptional repressor (16, 23). Interactions with postsplicing complexes have also been suggested (36), even though this finding may be controversial (33, 35, 42). How these various DEK functions in transcription, replication, and splicing relate mechanistically to carcinogenesis, autoimmune disease, or aspects of infectious viral life cycles is not understood.

Recent analyses of the transcriptome of senescing HeLa cervical cancer cells have reported an association of DEK repression with senescence. DEK overexpression could partially overcome the senescence phenotype in this system, emphasizing a functional role as a senescence inhibitor. Such a role is consistent with potential procarcinogenic activities for the human DEK oncogene. The replicative senescence of human primary foreskin fibroblasts and keratinocytes was also associated with DEK repression, and DEK overexpression in primary cells significantly extended their life span. High-risk but not low-risk HPV E7 protein induced DEK expression in primary HFKs, and together with studies of MEFs deleted for Rb family members, we have implicated members of the retinoblastoma family in transcriptional DEK repression. This is in agreement with a recent publication that has added to the original definition of the DEK promoter the finding that specific E2F binding sites are present and responsible for promoter activation by E2F family members 1, 2, and 3 (7, 45). The retinoblastoma protein is a critical mediator of senescence phenotypes (1, 43, 55) which functions, at least in some systems, through the stable repression of proliferation-associated E2F gene expression and irreversible cell cycle arrest (38). Reactivation of DEK in response to the mutational inactivation of Rb may therefore explain the observed upregulation of DEK mRNA levels in a number of human tumors, although potential functionally redundant roles for Rb family members as reported for MEFs (59) remain to be explored in these clinical settings.

Our data extend a role for DEK in senescence to apoptosis inhibition, which may similarly contribute to presumed procarcinogenic activities and may, at least in part, underlie the observed life span extension of primary cells following DEK overexpression (59). Given that p53 is a critical mediator of both senescence and apoptosis in many systems (34, 40, 51) and that DEK RNAi induced p53 activities in both cancer and primary cells (Fig. (Fig.7),7), we propose a model by which DEK upregulation may stimulate human carcinogenesis via inhibition of p53-driven tumor suppressor pathways. On the basis of our data depicted in Fig. Fig.8,8, we believe that p53 repression occurs at a posttranscriptional level and via increased protein turnover. Studies to examine the mechanism of p53 destabilization in detail are under way and might involve direct interactions between DEK and p53. These could also include proteins such as Hdm2, HdmX, or p14ARF that are already known to regulate p53 stability and degradation by the proteasome. Perhaps indicative of potential physical interactions between DEK and p53 complexes are structural similarities between a fragment of DEK comprising aa 78 to 208 and the SUMO ligase PIAS1, a protein that is known to bind p53 (39). Alternatively, p53 destabilization by DEK may be indirect and may be the result of DEK-chromatin interactions and subsequent signaling to specific regulators of p53 turnover. Such indirect mechanisms might be reflected in the relatively long time frame between DEK expression and p53 degradation (Fig. (Fig.8A).8A). Distinguishing between these possibilities in response to DEK overexpression and depletion will require detailed biochemical approaches, including definition of specific p53 modifications such as phosphorylation, sumoylation, and acetylation, as well as studies of a potential relationship between DEK-mediated chromatin modulation and the regulation of p53.

The fact that DEK RNAi-mediated apoptosis could be—at least partially—overcome via coexpression of the p53dd miniprotein implicates p53 transcriptional activity and the activation of downstream apoptosis inducers. Indeed, we have observed upregulated Bax protein levels in response to DEK depletion in primary cells (Fig. (Fig.7E),7E), and furthermore, preliminary experiments suggest that Bcl-X overexpression can inhibit DEKsh-induced cell death (data not shown). However, we cannot exclude the possibility of an additional role for nonclassical p53 functions at the mitochondria in the apoptotic phenotype, as well as p53-independent mechanisms of apoptosis induction. At least under some circumstances, reported studies of p53-mediated growth arrest versus apoptosis have emphasized levels of p53 protein and not necessarily its transcriptional activity in apoptosis phenotypes (8). Regardless, our model implies the existence of a regulatory loop between Rb and p53 via DEK, where relief of Rb-mediated DEK repression may result in inhibition of p53 activity. A regulatory loop that represents a barrier to uncontrolled proliferation has been firmly established between Rb and p53, in that Rb loss and increased E2F activity lead to ARF induction and consequent p53 stabilization (51). In this latter scenario, p53 apoptosis induction in response to Rb loss requires mechanisms to reinactivate p53 pathways in order to allow for clonal cancer cell outgrowth. Whether the generally aggressive nature of cancers that are associated with DEK induction represents a distinct cancer scenario in which the need to inactivate p53 is potentially lessened or circumvented remains to be investigated.

It is important to emphasize that while we have previously associated DEK repression with HeLa cell senescence, we describe here a full-fledged apoptotic phenotype in response to DEK RNAi. Definition of cellular pathways downstream from DEK that may influence apoptosis versus senescence, together with a more detailed understanding of p53 involvement in either case, will be an important task for the future. In the HeLa cell senescence system, repression of the high-risk HPV E6 and E7 oncogenes was mediated by the BPV E2 protein and resulted in reactivation of the p53 and pRB tumor suppressor pathways, cellular growth suppression, and senescence (18, 57). In this system, DEK repression was clearly observed but DEK protein levels remained detectable during senescence (59). In contrast, DEK RNAi resulted in DEK knockdown below the level of detection by Western blot analysis (Fig. (Fig.3).3). It is therefore possible that the degree of DEK repression influences the cellular decision toward senescence versus apoptosis. Indeed, we note that although apoptosis by far predominates the phenotype in DEK-depleted HFKs, we consistently detected cells with a distinctly flattened morphology reminiscent of senescence (data not shown). Given the observed p53 regulation by DEK, it is conceivable that the level of p53 activity will turn out to be a factor in the mode of cell death that is ultimately selected. Alternatively, the status of other genetic programs may be crucial in the context of DEK repression and could be reflected by the fact that DEK repression in the senescent HeLa cell system was accompanied by the regulation of approximately 700 genes (56). We have not defined the cellular transcriptome following DEK RNAi, but the potential transcriptional role(s) for DEK indicates that such gene expression changes may be detected. Identification of gene regulatory programs in response to DEK depletion and comparisons between DEK-depleted senescent versus apoptotic cells may yield important information about p53-driven or p53-independent pathways that distinguish the respective phenotypes.

The reported transcriptional upregulation of DEK in multiple human tumors, together with its cell death inhibitory activities, have raised the question of whether DEK might be targeted specifically in human tumor cells. However, our data indicate that, for the purpose of new cancer therapies, DEK depletion may represent a poor approach in the absence of additional measures. Potential dependence upon wild-type p53 and the observed apoptotic phenotypes in both cancer and primary cells following DEK RNAi—even though more pronounced in HeLa compared to primary cells—will likely cause significant toxicity in bystander cells. Furthermore, given the relative absence of a response in SAOS-2 and presumably other cancer cells with p53 mutated or deleted, treatment options through the targeting of DEK would only be applicable to tumors expressing the wild-type p53 protein, such as cervical tumors. A better understanding of molecular pathways that are affected by DEK may improve our prospects of manipulating DEK functions for the purpose of disease treatment and may result in a better understanding of the role of DEK as a cellular survival gene.


We thank Gerard Grosveld for the polyclonal DEK antiserum and James Lessard for the monoclonal actin antiserum. We thank Yang Shi for the BS/U6 empty and GFPsh vectors and Bert Vogelstein, Moshe Oren, Yi Sun, and Shiv Srivastava for providing p53-responsive reporter plasmids. We thank David Williams, Yi Zheng, James Mulloy, Harmut Geiger, and Erik Knudsen for helpful discussions and critical comments on the manuscript. We thank Teresa Cash for excellent technical assistance.

This research was supported by Public Health Service grants CA102357 and CA116316 from the National Cancer Institute to S.I.W.


[down-pointing small open triangle]Published ahead of print on 7 August 2006.


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