A functional genomics approach for the identification of putative tumor suppressor genes: Dickkopf-1 as suppressor of HeLa cell transformation

Andrei M. Mikheev1, Svetlana A. Mikheeva2, Binrong Liu3, Pinchas Cohen3 and Helmut Zarbl1,4

1 Program in Cancer Biology, Division of Public Health, Fred Hutchinson Cancer Research Center Seattle, WA 98104-2092, USA, 2 Department of Pediatrics, University of Washington Seattle, WA 98195, USA and 3 Department of Pediatrics, University of California, Los Angeles, CA 90095, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
We described previously the isolation and characterization of two non-tumorigenic revertants from the HeLa cervical carcinoma cell line, and demonstrated that loss of the transformed phenotype in these cells was the result of dominant somatic mutations. The goal of the present study was to use cDNA microarrays to identify candidate tumor suppressors among the set of genes whose increased expression correlated with loss of tumorigenicity in both revertants. Among the genes with significantly increased expression levels in both HA and HF revertants we identified Insulin Growth Factor Binding Protein-3 (IGFBP-3) and the Dickkopf-1 (DKK-1) genes. Both of these genes encode secreted proteins implicated in the modulation cell growth and differentiation, and IGFBP-3 was shown previously to have tumor suppressing activity. To test the hypothesis that increased expression of IGFBP-3 or the DKK-1 genes could have contributed to the suppression of tumorigenicity in the revertants, we expressed IGFBP-3 or DKK-1 in HeLa cells, and assessed their effects on anchorage dependent and independent growth, and tumor formation in athymic nude mice. Ectopic expression of IGFBP-3 or DKK-1 resulted in significantly decreased growth in soft agar. HeLa cells expressing ectopic IGFBP-3 or DKK-1 showed statistically significant differences in the kinetics of tumor formation. In any tumors that arose in animals injected with the IGFBP-3 expressing cells, there was a complete loss of IGFBP-3 activity, as measured by binding to IGF-1 and IGF-2 proteins. All tumors that arose after injection of cells expressing DKK-1, invariably showed almost a complete loss of ectopic DKK-1 expression. The observations that loss of DKK-1 expression or IGFBP-3 activity was required for tumorigenicity suggested that both proteins encode putative tumor suppressor genes. We also show that while DKK-1 expression does not affect cell growth in vitro, the protein does sensitize cells to apoptosis. We also demonstrated that effect of DKK-1 was not due to inhibition of ß-catenin/TCF4-regulated transcription. Taken together, our results indicate that somatic cell genetics combining with gene expression profiling may be a useful approach for the identification of functional suppressors of malignant cell growth.

Abbreviations: DKK-1, Dickkopf-1; DKKHA, HA-tagged DKK-1; IGFBP-3, insulin growth factor binding protein-3 gene; pOF, mutant reporter constructs; pOT, wild-type constructs


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Almost a decade ago we developed a highly efficient approach for the isolation of non-tumorigenic variants from populations of oncogene transformed cell lines or tumor cell lines (13). The approach was based on the findings of Lan Bo Chen (4), who made the observation that while all living cells actively take up the lipophilic cationic dye, rhodamine 123, retention of the dye within mitochrondria of many tumor cell lines is greatly extended relative to that of normal lines. We used fluorescence-activated cell sorting isolate mutant cells that had lost the prolonged dye retention phenotype, and then isolated individual clones of phenotypically normal cells that had lost their ability to form tumors in nude mice. Unlike previous methods for isolating revertants, this approach was rapid and efficient, and did not rely on prolonged in vitro selection protocols with agents that were themselves mutagenic. As a result, this somatic cell genetic approach was more likely to allow for the isolation of cells with a limited number of de novo mutations in genes that function as effectors or suppressors of cell transformation and tumorigenicity (13).

We used this approach previously to isolate independent, non-tumorigenic clones from the HeLa cervical carcinoma cell line (2,3). The revertants were generated by random mutagenesis of HeLa cells using ethylmethanesulfonate, followed by selection of the clones by FACS on the basis of their rhodamine 123 retention kinetics (1). Further phenotypic analyses demonstrated that unlike the parental HeLa cells, two of the revertant clones (designated HA and HF) were incapable of anchorage independent growth. Moreover, the revertants were not tumorigenic in the nude mouse assay. Both revertant clones also expressed reduced level of intestinal alkaline phosphatase, a cell surface marker, shown previously to correlate with HeLa cell tumorigenicity (5). Molecular characterization of the revertant cells revealed that both cell lines expressed increased steady state levels of p53 the tumor suppressor protein despite the continued expression of the HPV18 E6 and E7 viral oncogenes. Our preliminary findings indicated that the half-life of p53 protein in both revertants was prolonged relative to that measured in HeLa cells, consistent with post-translational stabilization of tumor suppressor in these cells. Studies by Goodwin and DiMaio (6) clearly demonstrated that complete restoration of p53 function in HeLa cells by exogenous expression of the HPV E2 gene, a transcriptional repressor of the E6 and E7 genes, resulted in the induction of p21-waf-1 and inhibition of HeLa growth. Despite increased levels of functional, wild-type p53 protein in the revertants, these cells failed to induce expression the p21-waf-1 inhibitor of cell cycle progression, a downstream effector of p53 mediated inhibition of cell growth. Consequently, we also observed a lack of G1 arrest following exposure of the revertants to DNA damaging agents (3). However, these finding could not rule out the possibility that partial reactivation of p53 detected in the revertants was sufficient to reverse the transformed phenotype of parental HeLa cells via a mechanism that did not involve p21 mediated inhibition of cell cycle progression. Although we initially proposed to identify the causal mutations using gene transfer experiments (7), this approach proved to be technically challenging and fraught with artifacts. As an alternative strategy, we combined our somatic cell genetics approach with genome wide expression profiling to identify candidate tumor suppressor genes, and then assessed their role in blocking tumorigenicity using functional in vitro and in vivo assays. To identify candidate genes comprising the p53 mediated, p21 independent tumor suppressor pathways, we used cDNA microarray technology to compare the gene expression profile of HeLa cells with those of the two independent revertant clones.

We reasoned that among the genes that are differentially expressed between the parental HeLa cells and both of the revertants, were genes comprising the biochemical pathways that contributed to the suppression of HeLa cell tumorigenicity. Among the candidate genes we identified two p53-regulated genes that both encode secreted peptides known to modulate cell growth and differentiation. The first of these, the insulin growth factor binding protein-3 gene (IGFBP-3), encodes a protein that sequesters the insulin growth factor to the extracellular matrix, and thereby prevents the tumorigenic growth of cancer cells expressing IGF. Among the set of genes up-regulated in both revertants, we also identified the Dickkopf-1 (DKK-1) gene, which also encodes a secreted protein implicated in inhibition of the Wnt signaling pathway. We then tested these genes individually for their ability to suppress the tumorigenicity of HeLa cells. Consistent with the hypothesis that elevated expression of p53-regulated modulators of cell growth can suppress tumorigenesis, ectopic expression of either IGFBP-3 or DKK-1 were incompatible with tumorigenic growth of HeLa cells in nude mice. The results also suggest that the combination of somatic cell genetics, genomics and functional assays may be a useful approach in identifying putative suppressor of tumorigenic growth.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Cells
HeLa cells (American Tissue Culture Collection, Rockville, MD) and revertant cell lines (HA and HF) were grown in DMEM supplemented with 10% calf serum (Hyclone, Logan, UT).

cDNA microarray and statistical analysis
Human cDNA microarrays comprising ~18 000 genes and expressed sequence tags were printed by Fred Hutchinson Cancer Research Center's DNA microarray facility. Information on microarray printing, hybridization and scanning is provided on http://www.fhcrc.org/shared_resources/genomics/. Total RNA was extracted from HeLa and revertant clones using RNAeasy RNA purification kit (Qiagen, Valencia, CA). Aliquots of 30 µg of total RNA were labeled by incorporating Amino-Allyl dUTP into cDNA using Reverse Transcriptase (Invitrogen, Carlsbad, CA), followed by coupling with Cy3 and Cy5 dyes (Amersham Biosciences, Chalfont St Giles, UK). A detailed description of labeling procedure can be found on the URL given above. Replicate hybridizations to microarrays were performed with independent RNA isolates, and were repeated twice for the HA clone and three times for the HF clone with a dye flip. RNA from parental HeLa cells was used as the reference for all hybridizations. Microarray slides were scanned at wavelengths of 532 and 635 nm using a dual-laser GenePix 4000A Microarray Scanner (Axon Instruments, Union City, CA), and fluorescence data were analyzed using GenePix Pro 3.0 software. Raw data were normalized assuming equivalent global hybridization of test and reference RNA to internal control spots consisting of genomic human DNA. Background levels were detected as signal intensity produced by non-specific DNAs present within each field on the slide. The average ratio of normalized intensities obtained from independent experiments, were used to evaluate expression levels of individual transcripts in revertant cells relative to HeLa cells. Genes with normalized intensities above background levels in at least one channel, and a ratio of intensities above 2 were considered differentially regulated. To ensure the statistical significance of data, only differentially regulated genes found in both revertant clones in at least two independent experiments of each revertant clone, are presented. Hierarchical clustering and data visualization was performed using Cluster and TreeView software packages (rana.lbl.gov/EisenSoftware.htm).

Plasmids
The IGFBP-3 expression vector (pKGBP3) was described previously (8). The IGFBP-3 expression construct was transfected in HeLa cells using Lipofectamine Plus reagent (Invitrogen) and resulting clones were isolated. DKK-1 cDNA was provided by Dr X.Chen (9). The C-terminal HA-tagged form of DKK-1 was generated by amplification with PCR primers (5'-CGGGATCCGCCACCATGATGGCTCTGGGCGCAG-3' and 5'-TTAA- GCGTAATCTGGAACATCGTATGGGTAGTGTCTCTGACAAGTGTGA-AGCC-3'). The resulting amplicon was subcloned in pCRII-TOPO vector and verified by sequencing. The BamHI fragment was then subcloned in the LXSH in the correct orientation. The untagged form of DKK-1 was created by cloning of the PCR amplified cDNA (5'-CGGAATTCATGATGGCTCTGGGCGCAG-3' and 5'-CGGAATTCTTAGTGTCTCTGACAAGTGTGA AGCC-3') into the EcoRI site of the LXSN expression vector. Following sequence verification, the resulting constructs were transfected into packaging Phoenix cells using Lipofectamine Plus reagent. After 24–36 h, media containing virus was collected, filtered and used for infection of HeLa cells in the presence of 4 µg/ml of polybrene (Sigma, St Louis, MO). Infected cells were selected by culturing cells in the presence of G418 or Hygromycin (Roche Applied Science, Indianapolis, IN) as appropriate. Pools and individual clones of hygromycin B or G418 resistant cells were collected and expanded, and ectopic expression of IGFBP-3 or DKK-1 was confirmed by northern or western blot analyses.

Soft agar growth
Soft agar growth was assessed by seeding 2 x 104 cells in DMEM with 10% of bovine serum and 0.33% molten Noble agar (Becton Dickinson, Sparks, MD). Assays were performed in duplicate and repeated three times. Colony formation was scored 3 or 4 weeks after seeding in soft agar.

Northern blot analysis
Total RNAs from cultured cells or tumors were extracted using Trizol reagent (Invitrogen), and dissolved in formamide (Fisher Scientific, Pittsburgh, PA). Between 12 and 25 µg of each RNA sample was loaded on a 1.1% agarose gel containing formaldehyde. Following electrophoresis, RNA was transferred to GeneScreen Plus (NEN Dupont, Boston, MA) nylon membrane by electroblotting for 2 h at 1 amp in 25 mM sodium phosphate buffer, pH 6.5, using a transblot apparatus (Bio-Rad, Hercules, CA). The IGFBP-3 (Incyte Genomics, St Louis, MO) and DKK-1 cDNA probes or the LXSH fragment containing hygromycin phosphotransferase gene were labeled by random priming with [{alpha}-32P]dCTP (NEN Life Science, Boston, MA). Each labeled probe was hybridized to a blotted membrane for 18 h in 1 M NaCl, 0.1 SDS, 1 µg/ml of calf thymus DNA (10). RNA loading was verified by the ethidium bromide staining of the blotted membrane or by hybridization to a labeled ß-actin cDNA probe.

Western and western ligand blot analysis
Western blot analysis was performed using 30 µg of whole cell lysate extracted with RIPA buffer supplemented with protease inhibitors. For detection of secreted proteins, 20 µl of filtered, dialyzed, conditioned media, collected after 48 h of incubation with subconfluent cells, was analyzed by western blotting. Following electrophoresis proteins were electro-blotted onto PVDF membranes (Millipore, Billerica, MA). Membranes were probed with IGFBP-3 purchased from DSL (Webster, TX) and subjected to purification on an IGFBP-3 column (11), HA-tag or actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) using standard procedures. Blotted antigens were detected using chemiluminescence reagent (NEN Life Science Products). For the western ligand blot, proteins were separated under non-reducing conditions. Following electroblotting, proteins were hybridized to radio-labeled IGF-1 and IGF-II as described previously (12).

Tumorigenicity in nude mice
All animal experiments were performed in FHCRC vivarium, which is fully accredited by AAALAC. All protocols used were reviewed and received the approval of the Institutional Animal Care and Use Committee. Female nude mice, 4 weeks old, were purchased from Harlan-Sprague–Dawley (Indianapolis, IN) and housed in filter-capped micro-isolation cages in temperature-controlled rooms maintained in a barrier facility on 12-h light/dark cycles and provided food and water ad libitum. Each mouse was injected subcutaneously with the indicated number of cells, and tumor growth was measured with calipers at weekly intervals, starting on the eleventh day after injection. On termination of the study, tumors were removed, trimmed of any connective tissue and frozen in liquid nitrogen. Tumor volumes were calculated using the formula: 0.4 x max. diameter x min. diameter2 (13). Statistical analysis of differences in tumor volumes and tumor frequencies were performed using Student's t-test and Fisher's exact test, as appropriate.

Anchorage-dependent growth assay
Cell cultures in log phase growth were harvested by trypsinization and counted using a Coulter counter (Coulter Electronics, Luton, UK). Aliquots of 105 cells were plated on 60 mm dishes in DME (Mediatech, Hemdon, VA) supplemented with 5% newborn calf serum in duplicate. Cells were harvested every 24 h for 3 days and total cell numbers were measured.

Apoptotic assay
Cells expressing DKK-1 or vector only were subjected to 10 mJ/cm2 of UV irradiation using UV chamber (Bio-Rad). Two hours later cells were harvested, stained with annexin V–Alexa Fluor 488 and propidium iodide (Molecular Probes, Eugene, OR) and subjected to Fluorescence Activated Cell Sorting. A total of 10 000 cells were counted per sample, and the data were processed using the FlowJo software (Tree Star, Ashland, OR).

Transient transfection assay
The reporter gene assay for ß-catenin/TCF-regulated transcription was performed using wild-type (pOT) and mutant reporter (pOF) constructs kindly provided by Dr B.Vogelstein and Dr K.W.Kinzler (14). Cells (1.5 x 105 cells seeded in 6 well plate) were co-transfected with reporter constructs (0.5 µg) and vector containing ß-galactosidase (0.2 µg) to normalize for differences in transfection efficiencies. Cells were harvested and disrupted by freezing/thawing. Luciferase and galactosidase activities were measured by standard procedures. Data are presented as the ratio pOT/pOF activities.

Immunofluorescence assay
Cells growing on the cover slips, were fixed in methanol/acetone (1:1) pre-chilled to -20°C. Following inhibition of non-specific binding in 1% BSA, cells were incubated with ß-catenin antibody for 1 h. Following extensive wash with PBS cells were incubated with secondary antibody conjugated with FITC (BD Biosciences Pharmingen, San Diego, CA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Differential expression of IGFBP-3 and DKK-1 in HeLa and revertant cell lines
We first sought to identify genes that are differentially expressed in both revertant clones when compared with the parental HeLa cells. We hypothesized that genes with increased expression levels in both of these independent revertant cell lines could be associated with molecular mechanisms contributing to the suppression of tumorigenicity of these cells. Gene expression profiles were obtained by individually co-hybridizing labeled cDNA generated using RNA from each revertant, with labeled cDNA generated with RNA from HeLa cells. Following extraction, normalization and statistical analysis of fluorescence intensity data, we identified 16 genes that were up-regulated, and 16 genes that were down-regulated in both revertants relative to parental HeLa cells (Table I). Among those reduced in both the HA and HF revertant cells were four genes involved in amino acid and protein metabolism. Also, down-regulated in both revertants was the gene encoding the extracellular matrix protein lumican, a member of the small leucine-rich proteoglycan family of proteins involved in cell migration and proliferation during embryonic development, tissue repair and tumor growth. Although reduced expression of lumican has been frequently reported in cervical and breast cancer (15,16), its role in suppressing the transformed phenotype of HeLa cells was not investigated in the present study. Disintegrin-like and metalloprotease with thrombospondin type 1 motif was shown to have angio-inhibitory activity (17).


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Table I. Genes differentially expressed in both HA and HF revertant clones relative to HeLa cellsa

 
Fibronectin, another extracellular matrix protein and ligand for integrin mediated cell adhesion and migration, embryogenesis, wound healing, blood coagulation, host defence and metastasis, was expressed at higher levels in the revertants as compared with HeLa cells. Paradoxically, previous studies have demonstrated that fibronectin expression is induced by HPV E6 and E7 oncoproteins (18,19). The unexpected observation that fibronectin expression is actually elevated in the HA and HF revertants may in part reflect the fact that the HPV E6 and E7 genes were not inactivated during generation of revertants (3).

Among the genes that were differentially expressed in both revertants were two signal transduction proteins shown previously to affect cell transformation. The first of these, Fibroblast Activation Protein (FAP), an integral membrane glycoprotein that belongs to the serine protease family, was down-regulated in both revertants. Over-expression of FAP in tumor cells was shown previously to potentiate tumor growth in scid mice (20). The second gene involved in signal transduction encodes the Protein Tyrosine Phosphatase gamma (PTPgamma) protein. Significantly, this putative tumor suppressor of kidney cancer was present at elevated levels in both revertants (21). Up-regulation of PTPgamma has also found in breast cancer cells undergoing terminal differentiation (22). The contribution of this gene in the phenotype of the revertants is under investigation.

The revertants also expressed elevated transcripts of two genes encoding secreted peptides shown previously to influence cell growth, differentiation and apoptosis. One of these IGFBP-3 was shown previously to suppress the tumorigenicity in certain cancer cells. The other transcript encodes the DKK-1 protein, originally identified for its role in normal head development in Xenopus and mice (23,24). DKK-1 mediates its effects by inhibiting Wnt signaling (25). Although there was no evidence to suggest that DKK-1 could suppress cell transformation, we selected DKK-1 and IGFBP-3 for further analysis for several reasons. First, both genes were expressed at very low levels in the parental cell line and were significantly elevated in both revertants. Moreover, previously studies indicated that the transcription of both genes was positively regulated by p53. Finally, both proteins are known to influence the kinase activity of glycogen synthase kinase-3 (GSK-3), a well studied effector of signaling that affects divergent pathways involved in metabolic control, development and cell transformation (26). Finally, IGFBP-3 was shown previously to suppress the tumorigenic growth of prostate cancer cells and lung carcinoma cells (see Discussion). Thus, in the present study, IGFBP-3 also served as a positive control for tumor suppressor gene identified using our functional genomics approach.

We first confirmed the differential expression of the DKK-1 and IGFBP-3 genes in HeLa cells and revertants using northern and western blot analyses. In HeLa cells, IGFBP-3 mRNA expression was close to the limit of detection by northern blot analysis and was dramatically increased in both revertants (Figure 1A). In all cells, the levels of IGFBP-3 protein expression correlated with levels of mRNA expression (Figure 1B). Expression of DKK-1 mRNA was undetectable in HeLa cells, and significantly elevated in both revertants, although the levels were higher in HF as compared with HA revertant cells (Figure 1A). Thus, the results from independent methods of analyses confirmed the differential expression data obtained using cDNA microarrays.



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Fig. 1. Elevated expression of endogenous IGFBP-3 and DKK-1 genes in revertant cell lines as compared with HeLa cells. (A) RNAs extracted from HeLa (H) and revertant cells HA (A) and HF (F) were subjected to northern blot analysis and probed with labeled cDNAs corresponding to IGFBP-3 and DKK-1. (Bottom panel) Ethidium bromide stained membrane following RNA transfer. (B) Proteins extracted from HeLa (H), HA (A) and HF (F) cells were subjected to western blot analysis and probed with IGFBP-3 antibody (top) and actin antibody (bottom).

 
IGFBP-3 and DKK-1 do not affect the rate of cell doubling
To assess the effect of these secreted proteins on HeLa cell growth and transformation, we used vectors that allowed for ectopic expression of the genes. Twelve independent clones expressing exogenous IGFBP-3 were isolated after transfection of HeLa with a previously described eucaryotic expression plasmid (8) and five representative clones are shown in Figure 2A. The DKK1 gene was transduced into HeLa cells using a retroviral vector. As no DKK1 antibodies were available and previous studies demonstrated that the addition of small peptides to N-terminal of the DKK-1 protein did not alter the function, we generated a retroviral expression construct which incorporated a HA-tag into the C-terminus to facilitate immuno-detection of the exogenous protein (DKKHA). Individual clones were isolated, and DKKHA protein expression levels were verified by western blot analysis. All clones that expressed the HA-tagged protein (Figure 2B) also secreted HA-tagged DKK-1 into the media (Figure 2B, right panel, only results for the clone randomly chosen for tumorigenicity studies are shown). Western blot analysis with the HA-tag antibody indicated that as is the case for the wild-type protein, the secreted HA-tagged DKK-1 was subjected to post-translational modification (23,25).



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Fig. 2. Effects of ectopic IGFBP-3 and DKK-1 expression on the anchorage dependent growth of HeLa cells. (A) Detection of secreted IGFBP-3 protein. Western blot analysis was performed on a 20 µl aliquot of filtered media conditioned for 48 h with subconfluent cultures of clones transfected with IGFBP-3 (clones 1, 2, 3, 4 and 8), vector transfected HeLa cells (V) and control media (S) before cell culturing (right panel only). (B) Independent clones were randomly isolated following infection of HeLa cells with a retroviral expression construct expressing the HA-tagged form of DKK-1 (numbers 1–5) or the control vector LXSH (V). HA-tagged DKK-1 protein (DKKHA) expression was verified by western blot analysis using an anti-HA antibody. Stripped membranes were subsequently hybridized to actin antibody to control for protein loading (bottom). (Right panel) Detection of secreted HA tagged DKK-1 in conditioned media. A 20 µl aliquot of filtered conditioned media from HeLa cells transduced with LXSH only or LXSH DKKHA [clone 3 from (A)] were probed with HA antibody. A comparison with the DKK-1 protein present in total cell lysate (CL) of the same cells demonstrated post-translational modification of the secreted form of HA-tagged DKK-1 protein (shown with double arrow). (C) A pool of positive clones expressing DKK-1 (left panel) or IGFBP-3 (right panel), and corresponding vector expressing cells (LXSH or pKG) were plated in 60 mm plates (105 cells/plate). The cells were harvested every 24 h for 3 days and counted. Data represent average numbers and standard deviation of three independent experiments performed in duplicate.

 
To determine if expression of DKK-1 or IGFBP-3 significantly altered cell growth kinetics in vitro, we seeded 105 cells (pools of individual clones) and monitored cell numbers every 24 h for 3 days (Figure 2C). The results indicated that cells expressing DKK-1 had a ~30% reduction in plating efficiency relative to the vector control cells after 24 h. Likewise, the IGFBP-3 expressing cells have a ~25% reduction in plating efficiency. However, further cell growth of DKK-1 expressing cells was not significantly affected. The average ratio of control to experimental cell numbers remained constant throughout the experiment (1.61 ± 0.3, 1.64 ± 0.19, 1.69 ± 0.24 for days 1, 2 and 3, respectively) indicating that DKK-1 expression did not affect significantly anchorage dependent cell growth. For IGFBP-3 expressing cells the average ratio of control to experimental cell numbers on days 1 and 2 were similar (1.37 ± 0.14 and 1.5 ± 0.28, respectively). On day 3 this ratio significantly increased (1.83 ± 0.22) compared with day 1 (P = 0.04). Thus, both IGFBP-3 and DKK1 had modest effects on the anchorage dependent HeLa cell growth compared with vector expressing cells.

IGFBP-3 and DKK-1 can individually inhibit anchorage independent growth of HeLa cells
Next we assessed the effect of IGFBP-3 and DKK-1 expression on anchorage independent growth. Despite clonal variation in the amount of secreted IGFBP-3 protein (Figure 2A), all IGFBP-3 expressing clones showed reduced, albeit variable, anchorage independent growth relative to vector controls (Figure 3A). Two representative clones, with either complete (clone 8) or partial (clone 4) inhibition of soft agar growth were chosen for further in vivo analysis.



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Fig. 3. (A) Ectopic expression of IGFBP-3 inhibits soft agar growth of HeLa cervical carcinoma cells. Soft agar growth of the same clones shown in Figure 2A shown at 4 weeks after seeding in 0.3% noble agar. The histogram shows the number of soft agar soft colonies formed by IGFBP-3 expressing clones (1, 2, 3, 4 and 8) as percent of vector transfected cells (V). Experiments were performed in duplicate and repeated three times. Shown is a representative experiment (not all clones tested shown). (B) Ectopic expression of the DKK-1 inhibits soft agar growth of HeLa cervical carcinoma cells. Soft agar growth of HeLa cells transfected with empty vector LXSH (shown in duplicate) or DKK-1 (only four clones shown). Pool of positive clones demonstrated similar inhibition of soft agar growth (not shown). Experiment was repeated three times in duplicate. Shown are representative cultures at 3 weeks after seeding in 0.3% noble agar. Histogram shows the number of soft agar colonies in DKK-1 expressing cells (clones) as a percent of vector expressing cells (V).

 
Ten clones that expressed exogenous DKKHA were also assayed for anchorage independent growth individually (five), and as a pool (all 10) (Figure 3B, only individual clones are shown). To conclusively rule out effects due to clonal variation and the HA-tag, we repeated the analysis with a pool of cells infected with a retrovirus expressing the untagged form of the DKK-1 protein. In all cases, cell expressing ectopic DKKHA or untagged DKK-1 showed dramatic reductions in both cloning efficiency and growth kinetics in soft agar medium relative to the vector controls (Figure 3B, only four clones shown). Together, these results demonstrated that ectopic over-expression of IGFBP-3 or DKK-1 genes resulted in significant inhibition of anchorage independent growth of HeLa cells, consistent with the hypothesis that the elevated expression of either or both of these genes contributed to the loss of anchorage independent growth of both revertant clones. We therefore tested the ability of IGFBP-3 and DKK-1 proteins to inhibit tumor formation in nude mice.

Tumorigenicity of HeLa cells expressing IGFBP-3 in nude mice
Previous studies demonstrated that IGFBP-3 can function as a tumor suppressor gene in some cell lines. Thus, IGFBP-3 served as a positive control in our in vivo tumorigenicity assay. Female athymic nude mice were subcutaneously injected with 107 cells expressing IGFBP-3, or the corresponding vector control cells. The results obtained with the two independent clones of HeLa cells expressing IGFBP-3 (clones 4 and 8 from Figure 2A) are shown in Figure 4A. Whereas tumors were detectable by day 11 in all animals injected with control HeLa cells, animals injected with clone 8 (BP3 c.8) failed to develop malignant tumors for up to 39 days after injection. Only one animal developed a small nodule that was barely palpable at the end of experiment.



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Fig. 4. Tumorigenicity of HeLa cells transfected with IGFBP-3 in nude mice correlates with loss of exogenous IGF binding activity compared with cells before injection. Incidence, in vivo growth kinetics and IGF binding activity were determined for nude mouse tumors induced by subcutaneous injection of HeLa cells with and without ectopic IGFBP-3 expression. (A) In vivo growth kinetics of two independent clones of HeLa cells transfected with IGFBP-3 (BP3 c.4, BP3 c.8), or with empty vector (pKGneo). All detectable nodules were measured once a week, and the results plotted as mean tumor volume ± SE at the time of measurement. Number of tumor bearing animals, defined as the total number of animal with any detectable nodule, shown in the table immediately below the graph. *Differences are statistically significant compared with tumors arising from HeLa cells transfected with the control vector (P < 0.05, actual P value is shown in the text). **For clone BP3 c.8, differences were statistically significant compared with control (P < 0.05) at every time point shown. (B) Expression of IGFBP-3 protein and IGF binding activity in tumors arising from BP3 c.4 cells, control tumors (vector) (only two controls are shown), and corresponding cells before injection. Proteins extracted from tumors and cells before injection were subjected to western blot analysis and probed with IGFBP-3 and actin antibodies. Parallel gel with the same amount of total proteins was electrophoresed under non-reducing conditions and hybridized to radio-labeled IGF1 and IGF2 (bottom, compare IGFBP-3 expressing cells before injection with tumors).

 
We also injected animals with IGFBP-3 expressing clone 4 (BP3 c.4), which demonstrated residual anchorage independent growth in soft agar (Figure 3A). Following injection of BP3 c.4 cells, tumor growth was significantly reduced at early times after injection relative to the vector control group. In the control group, tumors were detected in all animals (n = 4), and tumors had an average volume of 15.6 mm3 on day 11 after injection. During this time period, both mean of tumor volume and number of animals with detectable tumors, were significantly lower in animals injected with clone BP3 c.4 (P < 0.001 and P = 0.0143, respectively). At day 18 after injection, the mean of tumor volume generated by clone BP3 c.4 was still significantly lower when compared with the average tumor volume in the control group (P = 0.023). By day 25, the differences in the average tumor volume were no longer significant (P > 0.05), although the number of animals with detectable tumors were still lower than in the control group. Thereafter, there were no significant differences in tumor frequencies or volumes. Taken together, the data suggested that BP3 clone 4 was comprised of at least two clones, one expressing functional IGFBP-3 and a minor contaminant, possibly a spontaneous variant with an inactive transgene. Following injection into nude mice, the minor variant would be selected for its ability to grow in nude mice, albeit with delayed kinetics relative to controls.

Consistent with the latter hypothesis we demonstrated that none of the tumors that arose after injection of clone 4 retained the functional IGFBP-3 protein. Tumors arising from the injected BP3 clone 4 cells were assayed for both the levels of IGFBP-3 protein and IGF binding activity. We used western blot analyses to compare the levels of IGFBP-3 proteins in cells prior to injection with the levels in tumors. As shown in Figure 4B, cells from tumors showed a dramatic reduction in both IGFBP-3 protein levels and IGF binding activity relative to the cells before injection. Not only was there reduction in the total amount of ectopic IGFBP-3 protein (compare cells before injection with tumors), but in all tumors, the bulk of the IGFBP-3 antibody reactivity was associated with higher molecular weight forms of the protein. An IGFBP-3 reactive species of this molecular weight have not been reported in the literature. We confirmed that the higher molecular weight species of IGFBP-3 protein was not functional using western ligand blotting with labeled IGFs (Figure 4B, bottom panel). The results clearly demonstrated that there was little if any IGF binding activity in the tumors compared with the same cells before injection. Thus, selection for cells that were no longer capable of expressing the active form of IGFBP-3 protein capable of binding to IGFs was required for tumorigenic outgrowth. These results reinforced the finding obtained with IGFBP-3 expressing clone 8, providing clear evidence that suppression of the tumorigenic growth of HeLa cells in vivo required continuous expression of active IGFBP-3. Thus, our findings not only support the conclusion of other studies demonstrating that over-expression IGFBP-3 can inhibit the tumorigenicity, they also are the first to demonstrate that IGFBP-3 can inhibit tumorigenicity of cells transformed by HPV.

Tumorigenicity of HeLa cells expressing DKK-1 in nude mice
We next injected cells expressing either the HA-tagged DKK-1 (DKKHA clone 3) or the wild-type of DKK-1 (pool of clones) into nude mice (1). As was the case for cells expressing ectopic IGFBP-3, DKKHA cells showed statistically significant differences in the kinetics of tumor onset. On day 11 after injection, tumors were detected in all four control animals injected with the vector control cells, and attained an average volume 32.6 ± 6.3 mm3 (Figure 5A). At this same time point, there were no detectable tumors in any of the animals injected with DKKHA expressing HeLa cells (P = 0.002). Thereafter, the appearance of palpable tumors in DKKHA expressing clones occurred stochastically. Statistically significant differences in tumor volume between the control and DKKHA expressing cells remained between 18 and 25 days after injection (P = 0.029, P = 0.031, respectively). Only one mouse injected with DKKHA cells developed a palpable tumor by day 18 (P = 0.07) after injection. On day 25 after injection, the number of animals with measurable tumors arising from DKKHA expressing cells increased to three out of four animals. Significantly, by the end of the experiment, tumor volumes arising from the injected DKKHA expressing cells were significantly larger than those in animals receiving the vector control HeLa cells (Figure 5A, P = 0.04). These results were again consistent with the initial suppression of in vivo tumorigenicity by DKK-1, followed by selection for the outgrowth of cells with a higher tumorigenic capacity.



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Fig. 5. Tumorigenicity of HeLa cells transduced with HA-tagged form of DKK-1 in nude mice correlates with loss of exogenous DKK-1 expression. (A) Four animals in each group were injected with cells transduced with empty vector (LXSH) or HA-tagged DKK-1 (DKKHA). Results are presented as in Figure 4A. *Differences were significant compared with control animals (P < 0.05; actual P value shown in the text). (B) Total RNA was extracted from tumors generated following injection of HeLa cells transduced with empty vector (LXSH), HA-tagged form of DKK-1 (DKKHA) and corresponding cells before injection. Northern blots were hybridized to radiolabeled DKK-1 cDNA and actin cDNA. (C) RNA extracted from tumors (B) was subjected to northern blot analysis and hybridized to radiolabeled hygromycin phosphotransferase (Hygro) cDNA and actin cDNA. Similar results were obtained in independent experiment using pool of G418 resistant clones transduced with DKK-1 cloned in LXSN and injected in five nude mice (not shown for simplicity).

 
Using northern blot analyses, we compared the level of exogenous DKK-1 mRNA expression in cells before injection and the corresponding tumors. The results of this analysis clearly demonstrated that the cells in all tumors had significantly reduced expression of exogenous DKKHA relative to the levels in the cells before injection (Figure 5B), suggesting that tumorigenic outgrowth required loss of DKK-1 expression.

To rule out the possibility that lack of in vivo tumorigenicity was the result of clonal variation, we also infected HeLa cells with the retrovirus expressing the untagged DKK-1 cDNA. Since the vector also expressed the neomycin resistance genes, uninfected cells were eliminated by selection in G418. When this pool of cells expressing wild-type DKK-1 was injected into nude mice, we again observed a statistically significant delay in onset, followed by selective outgrowth of cells that did not express the exogenous DKK-1 in five of five animals by day 32 after injection (not shown). Thus, even after the injection of a pool of clones expressing DKK-1, we were unable to isolate tumors that retained expression of the exogenous DKK-1 gene. Taken together the results in two independent experiments using either individual clones or pool of clones, demonstrated that tumor growth following injection of HeLa cells, transduced with DKK-1, into nude mice was invariably associated with loss of DKK-1 expression.

To further explore the mechanism underlying the silencing of the exogenous DKK-1 gene in the tumor cells, we repeated the northern blot analyses analysis with a probe specific for hygromycin B resistance gene encoded by the retroviral vector used to transduce DKKHA. The results demonstrated that expression of the antibiotic resistance gene was also lost in all tumors arising after injection of DKKHA expressing cells. In contrast the hygromycin B resistance gene was expressed in tumors generated after injection of HeLa cells transduced with the control vector. The latter control confirmed that there was no selection against the expression of empty vector during the process of tumorigenesis (Figure 5C), rather, there was selection against a vector expressing the DKK-1 gene. As noted above, tumors that did develop after loss of the DKK-1 and hygromycin B resistance gene were significantly larger as compared with control tumors (P = 0.04). As cells were maintained in the presence of Hygromycin B prior to injection, it is unlikely that tumors arose from contaminating uninfected HeLa cells. Thus, it is reasonable to posit that inherent genomic instability of HeLa cells allowed for the rapid selection for clones of cells that have deleted exogenous DKK-1 and hygromycin B resistance gene driven by different promoters. Consistent with this supposition, Southern blot analyses using a vector specific hybridization probe indicated that in all tumors, the transducing retroviral sequences had undergone some form of clonal rearrangement resulting in reduction of molecular weight of vector specific sequence (not shown). Together these results unequivocally demonstrated that loss of DKK-1 expression was required for tumorigenic growth of these cells.

Expression of DKK-1 sensitizes HeLa cells to apoptosis
Previous studies demonstrated that the expression of IGFBP-3 or DKK-1 in tumor cells increases their sensitivity to apoptosis following a variety of treatments (11,24,27,28). Our unpublished studies have indicated that the revertants isolated from HeLa cells also have increased sensitivities to apoptosis. To determine if elevated DKK-1 expression can sensitize cells to apoptosis, we subjected a pool of DKK-1 expressing and control HeLa cells to UV irradiation. Two hours after irradiation, cells were harvested, and stained with annexin V conjugated with Alexa Fluor 488. FACS analysis showed that the number of annexin V positive (apoptotic) cells was increased 3.5-fold in DKK-1 expressing cells over non-irradiated controls (Figure 6). During the same time frame, the fraction of annexin V positive cells in vector expressing cells was increased only 1.8-fold compared with non-irradiated control. The percent of dead cells (cells stained with both annexin V and propidium iodide) was not significantly increased at 2 h after irradiation. These results indicated that DKK-1 expression affected an early stage of apoptosis following UV treatment.



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Fig. 6. DKK-1 expression sensitizes HeLa cells to apoptosis induced by UV irradiation. Control HeLa cells (LXSH) and HeLa cells expressing DKK-1 (pool of clones) were plated in duplicate (2.5 x 105 cells/60 mm dish). Cells were irradiated in UV chamber with 10 mJ/cm2 and harvested 2 h later. Cells were stained with annexin V and propidium iodide (PI) and subjected to FACS analysis. Histogram shows average percent (mean ± SE) of annexin V positive (apoptotic) and annexin V + PI positive (dead) cells before and after irradiation after three independent experiments. *Differences compared with UV irradiated control cells statistically significant (P < 0.05, two-tail t-test).

 
ß-Catenin/TCF independent effect of DKK-1 expression in HeLa cells
We next wanted to explore the mechanisms by which DKK-1 is able to suppress tumorigenic growth of HeLa cells. DKK-1 is a known inhibitor of the Wnt signaling pathway (see Discussion). Thus, the ability of DKK-1 to suppress the tumorigenicity of HeLa cell transformation suggested that these cells have a constitutively activated Wnt/ß-catenin signaling pathway. A hallmark for activation of the latter pathway is a dramatic enhancement in the activity of ß-catenin-regulated transcription through the activation of TCF/Lef-1, which then increases the expression of downstream targets, such as Cyclin D1, MMP-7 (matrilysin) and c-Myc. However, data from the microarray analyses indicated that the expression levels of these target genes were not altered in the revertant cells as compared with parental HeLa cells (Table II). These finding indicated that Wnt signaling was not deregulated in the revertants and that ectopic DKK-1 was probably not suppressing tumorigenicity by decreasing ß-catenin/TCF4 transcriptional activity. To further assess the activity of ß-catenin/TCF4-regulated transcription, independent clones (from Figure 5) and pool of clones expressing DKKHA, as well as a pool of cells expressing empty vector were transiently transfected with either the wild-type (pOT) or a mutant (pOF) TCF4 reporter construct. As positive control for ß-catenin mediated transcription, we used the SW480 colon carcinoma cell line, which has a constitutively activated ß-catenin/TCF4 signaling pathway (29). The ratio of pOT/pOF promoter activities were close to unity in control HeLa cells as well as in cells expressing DKKHA, which was significantly lower than the ratio in SW480 cells. These results indicated the levels of ß-catenin/TCF4-regulated transcription in HeLa cells were at background levels in cells with and without ectopic DKK-1 expression (Figure 7A). These findings were consistent with a previous study showing that activity of the cyclin D1 promoter was not affected by mutation in TCF-4 consensus binding sequence in HeLa cells, despite the fact that HeLa cells express TCF4 (30). The latter findings suggested that there is no activation of ß-catenin/TCF4 dependent transcriptional activity in HeLa cells. Taken together with the expression data from cDNA microarrays, these intriguing results suggested that inhibition of tumorigenicity by DKK-1 in HeLa cells might not involve inhibition of ß-catenin dependent transcription.


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Table II. Transcriptional activity of cyclin D1, MMP-7 and c-myc in revertant cells HA and HF compared with parental HeLa cells

 


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Fig. 7. (A) DKK-1 expression has no effect on ß-catenin/TCF4-regulated transcription in HeLa cells. SW480, HeLa cells transduced with empty vector or with the Ha-tagged DKK-1 (DKKHA) expression vector were transiently transfected with wild-type (pOT) and mutant reporter (pOF) constructs as described. Data presented as the ratio pOT/pOF promoter activities (mean ± SD). Results show individual clone of HeLa cells expressing DKKHA (clone) and pool of hygromycin B resistant clones. (B) Subcellular localization of ß-catenin is not affected by DKK-1 expression. HeLa cells transduced with empty vector (V) and DKK-1 were examined by immunofluorescence using an anti-ß-catenin antibody. HeLa cells processed without primary antibody (C) were used as negative control. Long arrows indicate membrane localization of ß-catenin, which was primarily detected in regions of cell-to-cell contacts. Wide arrows indicate perinuclear localization of ß-catenin.

 
To further confirm this conclusion, we next investigated the effects of DKK1 on the subcellular localization of ß-catenin using immunofluorescence assay. We found that in HeLa cells, ß-catenin is primarily associated with the membrane and the perinuclear region of the cytoplasm. As predicted from the low levels of TCF/Lef-1 activity, there was no significant accumulation of ß-catenin in the nuclei of the HeLa cells before or after ectopic expression of DKK1 (Figure 7B).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
In the present study we developed a functional genomics approach for the identification of genes involved in the functional suppression of cell transformation and tumorigenicity. The approach combines the robust method we developed previously for the rapid isolation of rare non-tumorigenic revertants from tumor cell populations (1), with cDNA microarray technology and traditional methods for the evaluation of tumorigenicity in vitro and in vivo. From among the genes that were up-regulated in two independent revertant cell lines, we selected two secreted proteins, IGFBP-3 and DKK-1, as candidate suppressors of the HPV-induced transformation and tumorigenicity of HeLa. One of these proteins (IGFBP-3) was implicated previously in the inhibition of tumorigenicity, and thus served as a positive control for the ability of this approach to detect genes with tumor suppressor activity, albeit not in HPV positive cells. IGFBP-3, the most abundant IGFBP in serum, plays a central role in inhibition of growth by sequestration of IGF-1 from its cognate receptor, thereby inhibiting anti-apoptotic and mitogenic activity of IGFs (31,32). Inhibition of IGF-1 receptor null fibroblasts by IGFBP-3 also suggested IGF-independent effects on cell growth (33), although a cell surface receptor for IGFBP-3 has not yet been identified. Expression of exogenous IGFBP-3 was shown to sensitize cells to irradiation-induced apoptosis (34). The latter effect is p53 independent as IGFBP-3 is active in p53 negative cells (11). IGFBP-3 also mediates the apoptotic actions of other agents such as vitamin D (35). Expression of IGFBP-3 in lung carcinomas or prostate carcinoma cell lines inhibited tumorigenicity in nude mice (3638). IGFBP-3 was shown to interact with retinoid X receptor-alpha and regulate downstream transcription and apoptosis (39). IGFBP-3 can signal through modulation of phosphorylation of Smad2 and Smad3 proteins, two known intermediates of the TGF-ß1 singling pathways (40).

Recent studies demonstrated that the E7 oncoprotein of HPV degrades IGFBP-3 via the ubiquitin–proteasome system (41). The latter observation suggested that IGFBP-3 could inhibit cervical cancer cell transformation, but only if expressed at sufficient levels to overcome HPV effects on its stability. The levels of IGFBP-3 expression attained in most of the IGFBP clones generated in the present study were sufficient to inhibit the soft agar and tumorigenicity of HeLa cells. Clone BP3 c.8 showed almost a complete inhibition of anchorage independent growth in vitro and of tumorigenicity in vivo. In contrast other clones such as BP3 c.4 showed incomplete inhibition of soft agar growth, which was correlated with the kinetics in vivo tumorigenicity. Although clone BP3 c.4 did grow in soft agar and in nude mice, tumor incidence occurred with delayed kinetics of onset. Furthermore, the delayed outgrowth of tumor growth was invariably associated with loss of IGFBP-3 activity, as measured by binding to radiolabeled IGFs. Thus, our experiments demonstrated that expression of IGFBP-3 activity is sufficient to inhibit tumorigenicity of HPV positive HeLa cells, and that loss of IGF binding activity is essential for tumorigenicity in these cells. Although inhibition of tumorigenicity of HeLa cells by IGFBP-3 over-expression was not unexpected, the results validated our functional genomics approach.

DKK-1, also a secreted protein, plays an important role in normal head development in Xenopus and mice (23,24). The five members of the Dickkopf family of the genes described to date differ in their structures, expression patterns, as well as their abilities to inhibit Wnt signaling (25). Although initially DKK-1 was identified as a p53 responsive gene (9), more recent studies indicated that induction of DKK-1 by DNA damage does not correlate with p53 status of the cells. Over-expression of DKK-1 sensitizes cells to apoptosis induced by ceramide in vitro affecting the BAX/BCL-2 expression ratio (28). The results obtained in the present study demonstrate that over-expression of DKK-1 inhibited tumorigenicity. HeLa cells with ectopic DKK-1 expression showed reduced anchorage independent growth in vitro and tumorigenicity in vivo. These latter effects were in part mediated by an increased sensitivity to apoptosis. After injecting in nude mice with HeLa cells expressing DKK-1 some tumors did develop, albeit with significantly delayed kinetics. Moreover, these tumors invariably arose from clones that no longer expressed the exogenous DKK-1 gene. DKK-1 signaling is mediated in part by the formation of a ternary complex with LRP6 and Kremen cell surface proteins, triggering the internalization and depletion of LRP6 co-receptor and inhibition of the canonical Wnt signaling pathway (4244). As a result GSK-3ß is activated and interacts with APC, axin, casein kinase 1{alpha} and ß-catenin. Resulting complex catalyses the phosphorylation of ß-catenin and its targeted degradation via the proteosome pathway (45). Degradation of ß-catenin is thought to inhibit activity of TCF/Lef transcription factor and transcription of downstream targets (c-myc, cyclin D, MMP-7) (46). Mutations in components of Wnt signaling pathway lead to cancer susceptibility and somatic mutations within Wnt pathway were frequently found in a wide variety of human cancers (for review see refs 47,48).

The inhibition of HeLa cell tumorigenicity by DKK-1 therefore suggested a possible role for the Wnt pathway in HPV induced cells transformation. Although the exact mechanism of the tumor suppressive effect of DKK-1 in HeLa cells remains to be determined, it does not appear to involve inhibition of the ß-catenin dependent transcription. We demonstrated that TCF/Lef transcriptional regulation by ectopic expression of DKK-1 was unchanged in HeLa cells, and was very low compared with SW480 cells. We also showed that in the revertant cells, expression levels of cyclin D1, MMP-7 and c-myc were not affected by DKK1. Another study (30) demonstrated that cyclin D1 promoter is not regulated by ß-catenin/TCF4 in HeLa cells. These observations are consistent with our results showing that DKK-1 mediated tumor suppressive effect was not mediated by inhibition of ß-catenin/TCF4 mediated transcription. The possibilities that DKK-1 mediates its tumor suppressive effect via other non-canonical components of Wnt pathway (Wnt/Ca2+, Rho) are under investigation.

Following the injection into nude mice, we observed strong biological selection for cells able to escape the growth inhibitory effects of IGFBP-3 and DKK-1. In vivo and in vitro selection for tumor cells with a more aggressive malignant phenotype is a well-documented phenomenon. Previous studies demonstrated the selection for cells with decreased responsiveness to TGF-ß in vivo (49). Hypoxia in fast growing tumors can contribute to the selection of tumors with a more transformed phenotype. For example, murine B16 melanoma cells subjected to sequential rounds of exposure to hypoxia and confluence in vitro produced populations with significantly enhanced growth capabilities, which could establish dominance within tumors (50). Assuming that secreted IGFBP-3 and DKK-1 proteins initially inhibited outgrowth of cells, in vivo selection would result in the outgrowth of cells that had lost or silenced expression of the transgenes. Analysis of tumors indicated that there was in fact in vivo selection against IGFBP-3 and DKK-1. As a control, we demonstrated that in vivo selection had no effect on the expression of the hygromycin B resistance gene, which provide no selective effect. The observed tumor incidence and kinetics of onset indicated that gene silencing or loss of these growth inhibitors in vivo was a stochastic event. However, the observation that all injected animals eventually developed tumors (except those for BP3 c.8, see Figure 4A) indicated that gene silencing or loss of activity occurred at a relatively high frequency. As the selection for cells with increased tumorigenic potential was independent of the transgene or the vector used to express transgene in the HeLa cells, it is probably that the inherent genomic instability of the parental HeLa cells contributed to random mutational events that were subsequently selected for in vivo. Such a model predicts that selection for loss of the transgenes would select for clones with even higher genomic instability. Consistent with this hypothesis, we observed that in vivo the selection for clones that eliminated the exogenous suppressor genes also resulted in tumors with a more aggressive growth phenotype when compared with the parental HeLa cells (Figure 5A).

The observation that tumors arose from variants that lost the ectopic tumor suppressors also provides mechanism insights. The outgrowth of the tumorigenic clones was not inhibited by the IGFBP-3 or DKK1 proteins secreted by the majority of the injected cells. Thus, either the tumorigenic variants were insensitive to paracrine effects of these secreted growth regulators, or outgrowth required in vivo elimination of the expressing cells by apoptosis. Although ectopic expression of IGFBP-3 or DKK1 reduced the cloning efficiency of HeLa cells in vitro, and increased their sensitivity to apoptosis following DNA damage, it is not clear that these effects are of sufficient magnitude to account for the in vivo selection of variants.

The HPV E6 and E7 oncogenes are thought to play a major role in cell senescence and transformation (51). The mechanism of HPV-induced cell transformation includes inactivation of p53 and Rb tumor suppressors by E6 and E7, respectively. Thus, the observation that ectopic expression of either IGFBP-3 or DKK-1 was able to inhibit tumorigenicity HeLa cells, predicts that these secreted proteins may also affect the tumorigenicity of human cancers harboring mutations in these tumor suppressor genes.

In summary, we have combined somatic cell genetics with functional genomics to identify the DKK-1 gene as a functional suppressor of cell transformation and tumorigenic growth, possibly via mechanisms independent of the canonical Wnt signaling pathway.


    Supplementary material
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Supplementary material can be found at: http://www.carcin.oupjournals.org


    Notes
 
4 To whom correspondence should be addressed Email: hzarbl{at}fhcrc.org Back


    Acknowledgments
 
We thank X.Zhang for assistance with animal experiments, and Drs L.Jing and H.Xie for statistical analyses, Dr X.Chen (Medical College of Georgia, Augusta) for providing DKK-1 cDNA, Dr B.Vogelstein and Dr K.W.Kinzler for providing the reporter construct for ß-catenin/TCF-regulated transcription. This study was supported by Public Health Services grants including the National Cancer Institute (1RO1CA47571, Helmut Zarbl, P.I.), the UW NIEHS sponsored Center for Ecogenetics and Environmental Health, NIEHS P30ES07033, David Eaton, P.I.), a Cancer Center Core Grant from the National Cancer Institute (CA 15704; Leland Hartwell, P.I.), as well as NIH grants: RO1AI40203, UO1CA84128, R01AG20954, DOD23026, and UO1CA92131to P.Cohen.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received May 8, 2003; revised September 11, 2003; accepted September 28, 2003.