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Cancer Research 67, 4742, May 15, 2007. doi: 10.1158/0008-5472.CAN-06-3610
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

The Ability of Versican to Simultaneously Cause Apoptotic Resistance and Sensitivity

David P. LaPierre1,2, Daniel Y. Lee1,2, Sen-Zhu Li3, Yi-Zhen Xie3, Ling Zhong1,2,4, Wang Sheng1,2, Zhaoqun Deng1,2 and Burton B. Yang1,2

1 Sunnybrook Health Sciences Centre and 2 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; 3 Guangdong Institute of Microbiology, Guangdong Academy of Sciences; and 4 School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China

Requests for reprints: Burton B. Yang, Sunnybrook Health Sciences Centre, Research Building, 2075 Bayview Avenue, Toronto, Canada M4N 3M5. Phone: 416-480-5874; Fax: 416-480-5737; E-mail: burton.yang{at}sri.utoronto.ca.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of the extracellular matrix proteoglycan versican is associated with more than 10 types of cancers, often being secreted by stromal cells in response to tumor signals. Previous work in our lab has shown that overexpression of the V1 versican isoform in cultured fibroblasts (V1 cells) increases both proliferation and apoptotic resistance. We show here that V1 cells induced tumor formation in nude mice and that, in keeping with previously shown apoptotic resistance, V1 cells have down-regulated Fas mRNA and protein levels. Unexpectedly, however, V1 cells were found to be sensitized to a wide range of cytotoxic agents. This combination of selective apoptotic resistance and sensitivity is often seen in cancer cells. V1 cells were also shown to have high resting levels of p53 and murine double minute-2 proteins, correlating with apoptotic sensitivity. Treatment with UV radiation induced p21 expression in vector-transfected cells but not in V1 cells. As p21 induces cell cycle arrest and inhibits apoptosis, its loss in V1 cells, coupled with high resting levels of proapoptotic p53, may be at least partially involved in their premature death following cytotoxic treatment. This study further supports the importance of versican in cancer cell biology and the complexity of apoptosis regulation. [Cancer Res 2007;67(10):4742–50]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Versican is a large chondroitin sulfate proteoglycan found in the extracellular matrix as four isoforms, V0, V1, V2, and V3, each with distinct structure, tissue localization, and signaling function. V0 and V1 are found during development and in tissues characterized by proliferation and matrix turnover in the adult (1). V0 and V1 are also expressed in hypertrophic scars and in the stroma of a wide variety of cancers. Studies of breast cancer have shown versican expression by fibroblasts in response to signals from carcinoma cells (2, 3). Increased stromal versican deposition is correlated with breast cancer relapse and prostate cancer progression (2, 3).

V1 versican can increase proliferation and activity in mitogenic signal transduction proteins epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK; ref. 4). Normally, potent checkpoints induce senescence or apoptosis to constrain the expansion of cells that have undergone oncogene activation and subsequent increases in proliferation (5). For a neoplastic clone to survive, it must acquire genetic or epigenetic lesions in its apoptotic signaling machinery (6). Accordingly, along with its effects on proliferative capacity, versican has also been shown to increase apoptotic resistance in a number of ways. Expression of the G1 and G3 domains of versican protects cells from apoptosis induced by cytotoxic drugs or death receptor ligands (7). G3-expressing cells have been shown to be resistant to apoptosis following treatment with hydrogen peroxide (8). Similarly, V1 versican–transfected cells show increased survival in serum-free conditions, contain decreased levels of the proapoptotic protein Bad, and are resistant to treatment with a proapoptotic drug targeting the mitochondria (4).

As cancer cells develop resistance to apoptosis, they almost uniformly become sensitized to specific apoptotic stimuli, such as chemotherapeutics, at the same time. This is largely due to accelerated proliferation and involves checkpoint proteins such as p53 (9). We therefore hypothesized that apoptotic resistance following V1 versican overexpression is accompanied by selective apoptotic sensitization. Whereas this study shows further apoptotic resistance in V1 versican–transfected cells through down-regulation of the death receptor Fas, these cells showed significant sensitivity to apoptosis induced by various cytotoxic agents. Resting levels of p53 were significantly up-regulated and downstream signaling by the antiapoptotic protein p21 was decreased in these cells. This study thus further shows the importance of versican in mediating apoptosis while also revealing the complexity of its effects.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. The NIH 3T3 murine fibroblast cell line was purchased from the American Type Tissue Collection (ATCC). DMEM and antibiotic-antimycotic solution was purchased from Wisent. Bovine calf serum was from Life Technologies, Inc. G418 was from Invitrogen. The enhanced chemiluminescent Western blotting detection kit was from Amersham. Fugene 6 transfection reagent was from Roche. The UV Stratalinker was from Stratagene. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit was from Molecular Probes. The RNeasy RNA column-based extraction kit and gel extraction kit were from Qiagen. The anti-Fas (rabbit), anti–murine double minute 2 (Mdm2; mouse), and anti-p21 (goat) antibodies were purchased from Santa Cruz Biotechnology. The anti-p53 (mouse) antibody was from Calbiochem. The anti-actin (mouse) antibody was from Sigma. All secondary antibodies, coupled to horseradish peroxidase, were from Bio-Rad. The caspase-3 assay kit was from Sigma. The Annexin V assay kit was from BD Biosciences. All chemicals were from Sigma.

Tumorigenicity in nude mice. Cell cultures at 80% confluence were harvested by trypsinization and washed thrice with serum-free DMEM, and viable cell numbers were determined using a hemocytometer. The cells were injected s.c. into 6-week-old strain CD1 nude mice using 1 x 107 cells in 150-µL DMEM per injection site. Animals were monitored weekly for tumor development over 6 weeks.

Construct generation. The full-length versican V1 and V2 constructs were generated as previously described (4). Mouse Fas cDNA (Image clone 30302649) was purchased from the ATCC in the pDNR-LIB vector. Fas cDNA was restricted using EcoRI and ApaI, and the resulting fragment was gel purified and ligated into a restricted pcDNA 3.1 vector (Invitrogen). Following ligation, the pcDNA 3.1-Fas construct was used to transform competent cells (Invitrogen) and colony screening was carried out using ampicillin-containing plates and gel verification.

To generate small interfering RNAs (siRNA) against p53 or Mdm2, two PCR primers were designed for each construct. The PCR product was digested with BglII and HindIII and inserted into the BglII- and HindIII-digested BluGFP, a vector with a BlueScript backbone, a cytomegalovirus promoter driving green fluorescent protein (GFP) expression, and a promoter driving the expression of the siRNA. This plasmid was developed in our lab and is expected to simultaneously silence targeting gene expression and produce GFP. This means that every fluorescent cell expresses the siRNA against p53 or Mdm2. The target sequence for p53 was 5'-ccacttgatggagagtatt-3'and for Mdm2 was 5'-aacgacacttacactatga-3'.

Induction of cell death. After cell counting, equal numbers of each cell type were plated the night before experiments. The medium was completely removed from the culture dishes and, working quickly, cells were placed in a UV Stratalinker at the same location each time. Cells were given various doses of UVC light (254 nm), and fresh medium was added back to the cells. Following defined time points, cells were analyzed as described. For Western blotting, reverse transcription-PCR (RT-PCR), and Annexin V assay, cells were plated onto 100-mm dishes. The procedure for Western blotting was the same as described (10). For light microscopy analysis, cells were plated onto 12-well plates. For the MTT assay, cells were plated onto 96-well plates. For each experiment, cells were plated to obtain 80% confluency the next day. For chemical treatment, the cells were plated the night before experiments. Before treatment, culture medium was changed followed by treatment with various doses of chemicals. For the caspase-3 assay, cells were plated and treated in 96-well plates according to the manufacturer's instructions, with fluorescence of caspase substrate measured with a FL600 fluorescence plate reader (Bio-Tek). Analyses of cell proliferation and apoptosis were done as described for the MTT assay (11) and the Annexin V apoptosis assay (8).

Cells were also seeded on 12-well tissue culture plates at a density of 1 x 105 per well in DMEM containing 10% serum. Ganoderma oil or conjugated linoleic acid, dissolved in DMSO, was added to the cultures at different concentrations. The DMSO vehicle served as a control. The cultures were maintained in a tissue incubator at 37°C containing 5% CO2 for 2 days. Cells were harvested and the cell number was determined with a Coulter counter as described (4, 12). Alternatively, dead cells in the medium were removed and the plates were washed followed by cell counting per image field with or without cell staining.

RT-PCR. Total RNA was extracted from vector- or versican V1–transfected cells 0, 4, or 8 h following treatment with 100 J/m2 UV irradiation. Adherent cells were treated and trypsinized, with cells in suspension collected as well. Total cells were centrifuged, washed, and frozen at –80°C. An RNeasy kit was used to lyse cells and precipitate total RNA according to instructions. Following RNA concentration analysis, 3 µg of total RNA were used in the Superscript II reverse transcription reaction using random primers and following the supplied protocol (Invitrogen). The PCR was next carried out with cDNA, with various cycle numbers used according to specific band intensities of given primer pairs and following annealing temperature optimization. The following primers (Invitrogen) were used for amplifying cDNA segments: FasNF (5'-cccggatccagagttcatactc-3') and FasCR (5'-cccgtcgactcactccagaca-3') for Fas cDNA; Actin121F (5'-ccggcatgtgcaaagccggct-3') and Actin360R (5'-gctcattgtagaaggtgtggt-3') for a fragment of ß-actin; p53N (5'-atgactgccatggaggagtcacag-3') and p53-290R (5'-tgagaagggacaaaagatgacagg-3') for a fragment of p53; Mdm2-292F (5'-tgcaagcacctcacagattcc-3') and Mdm2-479R (5'-acacaatgtgctgctgcttct-3') for part of Mdm2; p21-137F (5'-aacggtggaactttgacttcgt-3') and p21-456R (5'-caatctgcgcttggagtgatag-3') for a fragment of p21; and VerF (5'-aaagtcgactgttcctttcttgcaggt-3') and VerR (5'-aaaggatccgagcaagacacagagact-3') for a fragment of versican. Following RT-PCR amplification, products were analyzed by agarose gel electrophoresis.

Statistical analysis. Statistical significance was determined by ANOVA and the Neuman-Keuls post hoc test using GraphPad software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
V1 induces cell transformation and tumorigenesis. We have previously shown that V1 transfection enhances cell proliferation and survival in serum-free conditions (4) and alters cell morphology (10). In the current study, we assessed whether V1 was able to induce cell transformation by culturing a number of cell lines derived from NIH 3T3 fibroblasts in soft-agar gels. Only the V1-transfected cells formed colonies (Supplementary Fig. S1A), suggesting that V1 exhibited a transforming activity in NIH 3T3 cells. In tumorigenic experiments, nude mice were injected s.c. with NIH 3T3 cells transfected with different constructs. The HeLa cell line was used as a positive control, whereas NIH 3T3 fibroblasts, vector-transfected cells, and medium alone served as negative controls. Our experiments revealed that tumor formation occurred only in the V1-transfected cells, as no tumor formation was observed at the other injection sites (Table 1 ; Supplementary Fig. S1B).


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  		Table 1. Tumor formation in nude mice

 
Increased sensitivity of the V1-transfected cells to different treatments. Because the V1- and vector-transfected NIH 3T3 cells are of similar genetic background, we used them as a cell model to test sensitivity to a series of treatments. Etoposide is a clinical antineoplastic drug that induces double-strand breaks, which can lead to apoptosis largely dependent on p53 function (13). We found a profound sensitivity to etoposide in the V1-expressing cells (Fig. 1A ). Confirmation of V1 expression was carried out by RT-PCR and Western blot (Fig. 1B). In the lysate prepared from the V1-transfected cells, a fragment with size of 88 kDa was detected. This seemed to be a proteolytic product because it has been reported that versican is cleaved by proteases such as ADAMTS-1 and ADAMTS-4 (14). MTT assays showed that viability of the V1-transfected cells decreased markedly following a 24-h incubation with 20 µmol/L etoposide, whereas the vector-transfected cells remained largely unchanged (Fig. 1C). Viability of the V1-transfected cells was further decreased 48 h after treatment. Similar results were observed following treatment with cisplatin, a cytotoxic agent that induces apoptosis through DNA cross-linking. The V1-transfected cells exhibited increased sensitivity to 10 µmol/L cisplatin compared with the vector-transfected cells (Supplementary Fig. S2).


Figure 1
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  		Figure 1. Sensitivity of the V1-transfected cells to treatments with etoposide and UV irradiation. A, analysis by light microscopy revealed that treatment with doses of 10 or 60 µmol etoposide induced significant cell death in the V1-transfected cells 24 h following treatment. B, expression of the versican V1 construct was confirmed by RT-PCR (top) and Western blot (bottom). The major form (~250 kDa, closed arrow), a glycosylated form (>300 kDa, open arrow), and a proteolytic product (<100 kDa, broken arrow) of the V1 isoform were observed. C, cells were analyzed by an MTT assay following treatment with 20 µmol/L etoposide, which produced significantly increased death in the V1-transfected cells. D, cells were treated with 0 to 90 J/m2 and analyzed by MTT assays, 24 h following UV irradiation. The V1-transfected cells showed decreased cell survival over all doses, as compared with the vector-transfected control. Bars, SD. *, P < 0.01, versus control; **, P < 0.001, versus control.

 
UV radiation is known to have many proapoptotic effects on cells and is a potent inducer of p53 apoptotic activity (15). Morphologic examination under a light microscope indicated that there was significant cell death following irradiation of the V1-transfected cells at 100 J/m2, and that the response was much more pronounced than in the vector-transfected cells (Supplementary Fig. S3). Cell viability analysis by an MTT assay quantitatively showed a larger decrease of viability in the V1-transfected cells over a range of UV doses, as compared with the vector-transfected cells (Fig. 1D). These data suggest that the V1-transfected cells had a lower threshold for UV-induced death and also died with shorter latency.

Because hypoxic conditions can induce apoptosis through the p53-associated pathway (16), the cells were treated with the hypoxia mimetics cobalt chloride (CoCl2) and desferrioxamine. On addition of these compounds, the V1-transfected cells showed increased sensitivity resulting in cell death (Supplementary Fig. S4A), although hypoxia mimetics took longer to induce cell death than UV and chemotherapeutics (Supplementary Fig. S4B and C).

Conjugated linoleic acid is a natural fatty acid exhibiting inhibitory effects against multistage carcinogenesis (17). We tested whether conjugated linoleic acid had an effect on the V1- and vector-transfected NIH 3T3 cells and found that addition of conjugated linoleic acid significantly induced death of V1-transfected cells but not of vector-transfected cells (Supplementary Fig. S5).

More and more cancer patients are using herbal medicines as complementary treatment, and one widely used supplement is the mushroom Ganoderma lucidum. It has been recognized that the spores of G. lucidum are more potent than other formulations (18) because the spores contain unique components in their lipid fraction. We have extracted this lipid fraction, named Ganoderma oil, using high-pressure carbon dioxide. The effect of the Ganoderma oil was tested on the V1- and vector-transfected NIH 3T3 cells. We observed that Ganoderma oil significantly induced death of the V1-transfected cells but exhibited little effect on the vector-transfected cells (Supplementary Fig. S6).

Confirmation of cell apoptosis. To confirm that the loss of cells was in fact programmed cell death, one of the V1-transfected cell lines was treated with 100 J/m2 UV, 20 µmol/L etoposide, or 500 µmol/L CoCl2. Following cell lysis, caspase-3 activity was measured by assaying the cleavage of a fluorescent substrate. As shown in Fig. 2A to C , there was a significant increase in caspase-3 activity 24 h following UV and etoposide treatment and 30 h following CoCl2 treatment. To confirm these data, Annexin V analysis was carried out 18 h following UV irradiation and etoposide treatment and 24 h following addition of CoCl2. A significant proportion of cells became positive for Annexin V staining following treatment, confirming apoptosis of the cells (Fig. 2D).


Figure 2
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  		Figure 2. Caspase-3 and Annexin V assays confirm that cell death across treatments is apoptosis. The V1-transfected cells were treated with UV (A; 100 J/m2 UV treatment for 24 h), etoposide (B; 20 µmol for 24 h), and CoCl2 (C; 500 µmol for 30 h). Cell lysate was analyzed for levels of caspase-3 activity (fluorescence units). Cells were also analyzed after Annexin V and propidium iodide staining using flow cytometry. D, apoptosis of untreated cells and cells treated with 100 J/m2 UV for 24 h, 20 µmol etoposide for 24 h, and 500 µmol CoCl2 for 30 h. Bars, SD. **, P < 0.001, versus no treatment.

 
Down-regulation of Fas and up-regulation of p53 and Mdm2 in V1-transfected cells. We examined the molecular basis associated with apoptotic sensitivity to various treatments seen in the V1-transfected cells. The death receptor Fas was examined because it is an important mediator of multiple apoptotic pathways associated with survival (19). However, in keeping with previously shown apoptotic resistance, RT-PCR analysis showed a complete loss of Fas mRNA in V1-transfected 3T3 fibroblasts, with a concomitant loss of protein expression (Supplementary Fig. S7A and Fig. 3A ). To examine whether there were any defects in the posttranslational processing of Fas, murine Fas cDNA was cloned and transiently transfected into the V1-expressing cells. The strong expression of Fas suggested that transcriptional repression of the Fas gene is a primary cause of the loss of Fas.


Figure 3
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  		Figure 3. Loss of Fas expression and up-regulation of p53 and Mdm2 in versican V1–transfected cells. A, Western blot analysis exhibited loss of Fas protein in the three V1-transfected clones (top). This loss is specific to the V1 isoform, as V2-transfected cells seemed to have slightly up-regulated Fas levels (middle). Transient transfection of a Fas cDNA into the V1-transfected cells (V1-Fas) produced strong expression of Fas protein compared with vector transfection into the V1-transfected cells (V1-vector) or cells only transfected with empty vector (vector; bottom). B, immunoblotting analysis in resting cells showed that p53 levels were much higher in the V1-transfected cells than in the vector-transfected cells. C, up-regulation of p53 is specific to the V1 isoform, as the V2-transfected cells did not show any increase in p53 levels (top). Cells were treated with 15 µmol/L MG-132 for the time points indicated (bottom). p53 levels increased strongly in the V1-transfected cells, whereas levels increased more slowly in the vector-transfected cells. D, Western blot analysis exhibited higher Mdm2 expression in the V1-transfected cells than in the vector-transfected cells. A doublet appeared in the V1-transfected cells, which may correspond to isoforms of Mdm2.

 
Given the key role of the tumor suppressor p53 in responding to deregulated proliferation and transformation, levels of p53 were next evaluated. RT-PCR revealed no obvious differences between vector- and V1-transfected cells (Supplementary Fig. S7B). Immunoblotting of p53 protein, however, showed that the V1-expressing cells had much higher levels of p53 protein than the vector-transfected cells (Fig. 3B). This change in expression seemed to be specific for the V1 isoform because the V2-transfected cells did not show any accumulation of p53 protein (Fig. 3C).

The increased levels of p53 in the resting V1-transfected cells was interesting due to the fact that, in the absence of apoptotic signals, p53 is normally quickly degraded by the 26S proteasome following ubiquitination. It was initially suspected that there might have been some defects in the degradation machinery that maintains p53 protein at low levels, and it was possible that the V1-transfected cells had decreased activity of the E3 ligase Mdm2, which is responsible for ubiquitinating p53 and targeting it for degradation (20). As was the case for p53, RT-PCR showed no apparent change in Mdm2 mRNA levels between the vector- and V1-transfected cells (Supplementary Fig. S7C). However, the V1-transfected cells showed an up-regulation of Mdm2 protein, suggesting posttranscriptional regulation of Mdm2 (Fig. 3D).

Because elevated p53 levels did not seem to be due to a lack of Mdm2, it was theorized that p53 may have been protected from ubiquitination and degradation, either by posttranslational modification or inactivation of Mdm2. The vector- and V1-transfected cells were incubated with 15 µmol/L of MG-132, a potent inhibitor of the 26 S proteasome. As expected, p53 protein showed stabilization in the vector-transfected cells, but this took ~6 h to occur (Fig. 3C). The V1-transfected cells also showed stabilization of p53 over time following proteasome inhibition, and this effect was much more pronounced than in the vector-transfected cells. These data suggest that p53 was indeed being degraded in the V1-transfected cells and that Mdm2 was functional in these cells. How p53 protein levels remain elevated in the V1-transfected cells despite concurrent Mdm2 up-regulation remains unclear.

Downstream effects of UV treatment on the V1-transfected cells. As the proapoptotic response to UV usually involves p53 stabilization and transcription of downstream cell cycle arrest and proapoptosis genes, immunoblotting of p53 following irradiation was carried out. p53 protein levels increased over time after exposure of vector-transfected cells to various doses of UV (Fig. 4A ), both peaking and then decreasing. Surprisingly, however, V1 cells exposed to these same doses of UV did not show any increase in p53 levels, suggesting that no further stabilization of p53 was occurring (Fig. 4A). Western blotting with lower amounts of protein loaded failed to reveal even slight increases in the V1-transfected cells (data not shown). It is of note that the increase of p53 in the vector-transfected cells, sufficient to induce apoptosis, did not approach levels seen in the V1-transfected cells.


Figure 4
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  		Figure 4. Altered gene expression in the V1-transfected cells following UV irradiation. A, cells were treated with 100 J/m2 of UV irradiation and were lysed 0, 4, or 8 h following treatment. Western blotting revealed that p53 levels increased and then decreased in the vector-transfected cells, whereas levels remained high and unchanged in the V1-transfected cells. Both cell types showed a decrease at 4 h in Mdm2 levels following treatment, and the vector-transfected cells had an elevation at 8 h. p21 was elevated in the vector-transfected cells at both 4 and 8 h, whereas no evidence of p21 protein was seen in the V1-transfected cells at any time point. Fas seemed to slightly increase in the vector-transfected cells and in one of the V1-transfected cell lines. B, cells were lysed 0, 4, or 8 h following treatment with 100 J/m2 UV irradiation, and RT-PCR analysis was carried out on the vector- and V1-transfected cells. As expected, p21 mRNA was up-regulated following treatment in the vector-transfected cells. However, no p21 was detected in the V1-transfected cells. Fas mRNA seemed to be slightly up-regulated in the vector-transfected cells, whereas there was no evidence of Fas transcription in the V1-transfected cells. Under these conditions, neither cell type showed any increase in Mdm2 levels. C, cell lysate prepared from NIH 3T3 cells transiently transfected with an siRNA construct against p53 was analyzed by Western blot probed with an anti-p53 antibody, confirming reduction in p53 expression. D, V1-expressing cells were transiently transfected without (control) or with siRNA constructs targeting p53 or Mdm2 or an empty vector expressing GFP alone, followed by UV treatment at 100 J/m2 for 0 (to analyze transfection efficiency), 1, or 2 d. Cells were analyzed with flow cytometry. An increase in the percentage of fluorescent cells meant resistance to UV-induced cell death. Typical flow cytometry results were shown.

 
Mdm2, the stability of which is intimately linked with p53 levels, participates in a negative feedback loop with p53. Transcription of Mdm2 is increased following p53 stabilization and acts to decrease p53 levels. This is preceded, however, by a transient decrease in Mdm2, which is p53 independent (21). These changes were observed in the vector-transfected cells following irradiation, with a decrease in Mdm2 protein levels observed at 4 h and with a slight increase 8 h after treatment. In the V1-transfected cells, there was a precipitous drop in Mdm2 levels 4 h posttreatment, with high levels of Mdm2 observed at 8 h (Fig. 4A). However, there did not seem to be any increase above resting levels at this last time point, as was observed in the vector-transfected cells. No observable changes were seen by RT-PCR analysis in either the vector- or V1-transfected cells following UV radiation (Fig. 4B).

p21, a cyclin-dependent kinase (Cdk) inhibitor that mediates G1 arrest, is an important transcriptional target of stabilized p53 (22). Immunoblotting of the vector-transfected cells exhibited an increase in p21 levels following UV exposure, whereas the V1-transfected cells failed to produce any p21 response (Fig. 4A). This result is interesting when considered along with our previously published results showing increased degradation and thereby lower levels of p27, another Cdk inhibitor, in V1 cells (4). Analysis of mRNA levels indicated that the vector-transfected cells produced a robust up-regulation of p21 mRNA, which correlated with increased stability and putative transcriptional activity of p53 protein (Fig. 4B). There was, however, no evidence of p21 transcriptional activity in the V1-transfected cells.

Fas has also been shown to be a transcriptional target of p53, and increased p53-mediated Fas expression is often correlated with DNA damage-induced apoptosis in a cell type-specific manner (23). Fas protein levels seemed to somewhat increase in the vector-transfected cells at 8 h, and there also seemed to be a slight increase in Fas levels in one of the V1-transfected clones (Fig. 4A). However, levels did not approach those seen in the vector-transfected cells.

To confirm that the increases in p53 and Mdm2 were responsible for the V1-transfected cell death, siRNAs targeting p53 and Mdm2 were generated. As p53 expression was greatly up-regulated in the V1-transfected cells, transient transfection of siRNA targeting p53 was able to produce a detectable reduction in the p53 protein level as analyzed by Western blot (Fig. 4C), although transfection efficiency was ~20% to 25% (Fig. 4D). Reduction in p53 and Mdm2 expression was also confirmed by immunocytometry analysis (Supplementary Fig. S8). The V1-expressing NIH 3T3 cells were transiently transfected with or without siRNA constructs targeting p53 or Mdm2. An empty vector expressing GFP served as a negative control, followed by UV radiation treatment at 100 J/m2 for 1 or 2 days. Cells were examined under a light and fluorescent microscope. It was observed that transfection with siRNA targeting p53 or Mdm2 led to increased percentages of fluorescent cells following UV treatment. This experiment was repeated, and analysis with flow cytometry confirmed this observation (Fig. 4D). These results indicate that transfection with siRNA constructs targeting p53 or Mdm2 rendered cells resistant to UV-induced cell death.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results presented here further describe the importance of V1 versican in tumor biology by showing that V1-transfected cells developed colonies in soft agarose gel and induced tumors in nude mice, whereas cells transfected with the V2 isoform did not. As previously shown, versican can lead to reduced levels of proapoptotic molecules such as Bad and decreased responses to apoptotic stimuli (4, 8). This study supports those results by showing a loss of Fas mRNA and protein expression. Fas can be down-regulated through active transcriptional repression and has been shown to be epigenetically silenced in cancer cells through DNA methylation (24).

Deregulated proliferation, occurring in tumor cells, has been well documented as a potent apoptosis inducer. Similarly, the V1-transfected cells examined here show increased proliferation. At a molecular level, these cells show high levels of EGFR expression and downstream ERK activation (4). There may be selective pressure for the loss of molecules such as Fas as a developing clone encounters tumor suppressor pathways designed to prevent its expansion. It is possible that signals common to neoplastic development and V1 overexpression result in reduced sensitivity to specific proapoptotic pathways to permit survival.

Whereas V1-transfected cells have shown resistance to apoptosis, they also have become significantly sensitized to other apoptotic stimuli, including UV radiation, chemotherapeutics, hypoxia mimetics, Ganoderma oil, and conjugated linoleic acid. Mechanisms behind the apoptotic sensitivity shown in this study have been outlined in Fig. 5 . V1-transfected cells have elevated resting levels of the tumor suppressor p53, which plays a key role in inducing apoptosis in response to various detrimental events, including DNA damage, hypoxia, and telomere erosion. It has also emerged as a key apoptotic inducer following oncogenic activation and, given its significance, is thought to be mutated in at least 50% of human tumors. However, increased levels of wild-type p53 do not guarantee cell cycle arrest, senescence, or apoptosis. Many cancers show high levels of p53 expression in the absence of mutations (25), and p53 expression is increased during the hair follicle growth cycle without inducing apoptosis during unstressed conditions (26). p53 levels are also up-regulated during wound healing (27).


Figure 5
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  		Figure 5. A model of changes in apoptotic sensitivity caused by V1 overexpression. A, cytotoxic agents such as UV can induce either cell cycle arrest or apoptosis, in part depending on the gating response of p21 downstream of p53. B, our results suggest that V1 versican causes proliferation, selective apoptotic resistance, and selective apoptotic sensitization. Apoptotic resistance has been shown here and in previous work from our lab. Sensitivity to cytotoxic agents shown here seems to be mediated, at least in part, by constitutive overexpression of p53 and a loss of p21 expression following UV treatment.

 
Elevated levels of p53 seem to have primed V1-transfected cells to cross the apoptotic threshold when confronted by proapoptotic challenges, bypassing cell cycle arrest. siRNA knockdown of p53 in V1-expressing cells led to increased survival following treatment, supporting the role of p53 in apoptosis in these cells. This suggests that the downstream signaling capacity of p53 has been altered, and indeed changes in p53 transcriptional activity in V1-transfected cells were shown by a lack of p21 induction following DNA damage. p21 both induces cell cycle arrest and inhibits apoptosis, thus playing an important gating function of the p53 response (28). The lack of p21 transcription in V1-transfected cells therefore seems to release inhibition of apoptosis and allow apoptotic induction by p53 following cytotoxic treatment.

Just as oncogenic activation and their resulting increases in proliferation are detrimental, transient increases in proliferation can be greatly beneficial. Hypertrophic wounds and cycling hair follicles are instances in which proliferation-induced apoptosis is avoided. Interestingly, versican expression is strongly increased in each of these locations (29, 30). The shown antiapoptotic effects of versican may therefore be at least partially responsible for cell survival as a component of the microenvironment. As mentioned above, p53 up-regulation in settings of increased cell cycle activity is also well documented, and the dramatic increase in p53 levels in V1-transfected cells is thus unsurprising. Although it is currently unknown how this has occurred, a well-described pathway linking increased proliferation to p53 stabilization involves p19ARF, which acts by disrupting the Mdm2-p53 interaction. However, NIH 3T3 cells have a biallelic mutation for the p19ARF locus (31). The increased expression of p53 presented here suggests that there remains another mediator of p53 stability that may become active following V1 overexpression. Proteins other than p19ARF have already been suggested to mediate proliferation-induced p53 responses. Using mechanisms that do not require p19ARF, oncogenic activation has been shown to potentiate the cell to specific apoptotic insults through p53 (32).

The response of the V1-transfected cells to genotoxic treatments is similar to that of testicular germ cell tumors and their extreme sensitivity to cisplatin, etoposide, and other chemotherapeutics. Strikingly, these tumors frequently express high levels of both versican and functionally inactive p53, which become operational following DNA damage (33, 34).

This study further confirms the specificity of versican isoforms in affecting cell behavior, in keeping with a large body of findings from our lab and various others. V1 versican induced cell transformation following transfection, whereas V2 did not. Likewise, changes in Fas and p53 levels and increased apoptotic sensitivity were preferentially seen in V1-transfected cells. V0 and V1 are associated with the proliferative phenotype, whereas V2 has been shown to cause antiregenerative effects in the central nervous system (35, 36). Work previously done on versican-expressing fibroblasts has contributed to these findings, showing that proliferative and antiapoptotic effects were specific to the versican V1 isoform, whereas V2 isoform transfection often resulted in opposite effects (4). The fact that V0 and V1 are preferentially seen accompanying cancer development lends support to the idea that the results seen here are relevant to in vivo findings (37, 38).

The microenvironment is growing in significance as a mediator of tumor progression, and the results of this study highlight the role of versican in affecting apoptosis. Many changes have occurred in the sensitivity of these cells to apoptosis, and this study underscores the complexity of cell death. Analysis of the signaling pathways linking versican to these changes should be of benefit to our understanding of apoptosis while further explaining the role of the tumor microenvironment in mediating cancer cell phenotype.


   Acknowledgments
 
Grant support: Canadian Institutes of Health Research grants MOP-62729 and MOP-74469 (B.B. Yang), a Career Investigator Award (CI 5958) from the Heart and Stroke Foundation of Ontario (B.B. Yang), and a Scholarship from National Sciences and Engineering Research Council (D.P. LaPierre).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Chung-Kwun Amy Wong for help in the preparation of the manuscript.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Current address for D.P. LaPierre: Faculty of Medicine, Dalhousie University, Halifax, Canada.

Received 9/28/06. Revised 2/22/07. Accepted 3/ 7/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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