Osteopontin induction is required for tumor promoter-induced transformation of preneoplastic mouse cells

Pi-Ling Chang1,2,4, Minhton Cao3 and Patricia Hicks1

1 Department of Nutrition Sciences and 2 Comprehensive Cancer Center, 311 Susan Mott Webb Nutrition Sciences Building, 1675 University Boulevard, University of Alabama at Birmingham, Birmingham, AL 35295-3360, USA and 3 School of Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294-6630, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Osteopontin (OPN) is a secreted, adhesive glycoprotein. Elevated expression of OPN in malignant and benign tumors is postulated to play a role in tumorigenesis. To determine whether OPN induction is required for tumor promotion, we used the in vitro JB6 model known to correlate with tumor promotion in vivo. The skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces irreversible transformation of JB6 Cl41.5a cells. Concomitantly, TPA markedly stimulates early and persistent OPN expression and secretion for at least 4 days (the time required for these cells to begin to acquire the transformed phenotype) and increases cells' adhesion to OPN. Here, we demonstrated that dexamethasone, a synthetic analog of glucocorticoid, known to inhibit tumor promotion in vivo, not only suppressed TPA-induced OPN mRNA expression and inhibited tumorigenic transformation of JB6 Cl41.5a cells (as previously shown in JB6 Cl22 and Cl41 cells), but also that the addition of OPN partially restored dexamethasone suppression of TPA-induced cell transformation. Therefore, we tested the hypothesis that OPN induction is required for tumor promoter-induced transformation of JB6 cells by examining (i) whether the addition of OPN will induce transformation, (ii) whether antisense OPN expression will inhibit TPA-induced transformation and (iii) if the latter experiment showed inhibition of TPA-induced transformation whether the addition of OPN will rescue this effect. Results indicated that the addition of purified OPN induced a dose-dependent transformation of JB6 cells, as assessed by anchorage- independent growth assay and that this induction was suppressed by antibody to OPN. Furthermore, antisense OPN expressing JB6 clones suppressed TPA-induced OPN synthesis and secretion and inhibited TPA-induced anchorage-independent growth, which was partially rescued by the addition of OPN. In conclusion, OPN induction is required and can be sufficient to induce in vitro cellular transformation of a preneoplastic murine JB6 cell line.

Abbreviations: FN, fibronectin; mOPN, mouse OPN; OPN, osteopontin; rOPN, rat OPN; TPA, 12-O-tetradecanoylphorbol-13-acetate


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The molecular mechanisms responsible for tumor promotion, the rate-limiting step in carcinogenesis, are complex and still unclear. Identifying molecular events required for traversing rate-limiting stages of multistage carcinogenesis is important for targeted cancer prevention or intervention. Cellular markers such as proto-oncogenes and tumor suppressors have been shown to play significant roles in facilitating the process of carcinogenesis. However, emerging evidence suggests that alteration in the microenvironment of the cells in terms of changes in the interaction of matrix proteins with their cell surface receptors can play a critical role in tumorigenesis and metastasis (14). Here, we determined whether an induced matrix protein osteopontin (OPN) is required for tumor promoter-induced tumorigenic transformation in vitro.

OPN is an acidic, adhesive, matrix glycoprotein. The secreted OPN possesses calcium-binding, chemotaxis, cell adhesion and cell signaling properties and is, therefore, postulated to play several functions (5,6), one of which is tumorigenesis (711). Elevated levels of plasma or serum OPN are detected in cancer patients (1216) and its expression found in cancer of varied tissue types (1721). Furthermore, high OPN expression in several benign tumors suggests that it could be induced during tumor formation and may play a role in tumor promotion (22,23).

The two-stage mouse skin carcinogenesis protocol, a model used for tumor promotion studies in vivo, consists of topical application of the carcinogen, 7,12-dimethylbenz[a]anthracene, followed by weekly application of the skin tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). This procedure results in the formation of papillomas and eventually squamous cells carcinomas of the skin. TPA has been shown to stimulate early expression of the steady-state OPN mRNA in the mouse epidermis and subsequently in papillomas and squamous cell carcinomas (24,25). These studies further implicate the possible role of induced OPN in tumorigenesis. High secretion of induced-OPN could by an autocrine or paracrine route adhere to cell surface receptors and impart intracellular signals to prevent apoptosis or promote clonal formation of initiated cells leading to papilloma formation and subsequently, squamous cell carcinomas. OPN has several adhesive amino acid sequences shown to interact with cell surface receptors such as integrins (11,2633) and CD44 (34,35). One of the better-known adhesive sequences in OPN is the argininylglycylaspartic acid-(RGD-)cell-binding motif, which interacts with {alpha}vß1, {alpha}vß3 and {alpha}vß5 integrins (11,2629).

In addition to in vivo studies, results from in vitro studies also support OPN's role in tumorigenesis. OPN expression is elevated in several malignant cell lines (8) and Ras-transformed NIH 3T3 cells (36). Suppression of OPN expression by antisense OPN or targeted ribozyme in metastatic cell lines showed not only reduced metastasis, but also reduced tumorigenicity and low colony formation in soft agar (3739) suggesting the potential role of OPN in tumor promotion. Since OPN expression has been shown to be regulated by a ras-activated enhancer (40,41), studies using OPN-deficient ras-transformed cell lines were established to determine their ability to transform in vitro and in vivo. These cell lines show reduced colony formation in soft agar. Furthermore, cells injected subcutaneously into nude mice develop tumors more slowly compared with wild-type cell lines, thus maximal transformation by ras may require OPN expression (9). Interestingly, crossing OPN-deficient mice (42) with v-Ha-ras/c-myc transgenic mice fails to show a decrease in tumor formation (10).

Although, most findings mentioned above implicate that OPN induction or high expression is involved in tumorigenesis, these studies utilize malignant cell lines or the addition of oncogene, which introduces promoting/transforming factor, and therefore, dissecting the role contributed solely by OPN is difficult. To isolate clearly the effect of OPN from that of oncogenic effects, we choose to use a non-malignant cell line such as the promotable JB6 cells, which lacks both mutated p53 and ras genes (43), to determine the requirement of OPN induction in tumor promotion. The JB6 model is the only cell culture model available that is predictive for events required in tumor promotion in vivo (4448). These mouse epidermal JB6 cells do not form colonies in soft agar nor tumors in nude mice (49). However, TPA treatment of the promotable JB6 clones Cl41.5a and Cl22 irreversibly transform these cells and concomitantly, stimulate a marked increase in OPN mRNA (50) and its protein expression and secretion (5153). Since these promotable JB6 cells require at least 4 days of TPA treatment to begin to develop a transformed phenotype (54), the early expression of TPA-induced OPN mRNA by 2–4 h, which persists for at least 4 days (55), suggest that OPN induction may contribute to the process of tumor promotion. Moreover, transforming TPA-treated JB6 cells show increased adhesion to OPN, which is mediated through its RGD cell-binding region and, most probably, the activated cell surface integrin {alpha}vß5 (11). Therefore, TPA-induced secretion of OPN may assist in promoting JB6 cell transformation by an autocrine pathway.

To determine whether induced OPN is an important player downstream of tumor promoter-induced transformation, we postulated that inhibitors of tumor promotion in vivo such as dexamethasone, a synthetic glucocorticoid (5659), should not only suppress tumor promoter-induced expression of OPN and tumorigenic transformation of JB6 Cl41.5a cells (as shown previously in other promotable clones JB6 Cl22 and JB6 Cl41, respectively; 44,60), but that the addition of OPN should rescue the attenuated cell transformation. Also, to specifically test the hypothesis that OPN induction is required for tumor promoter-induced transformation of JB6 cells, we examined (i) whether the addition of OPN (over-expression) promotes tumorigenic transformation, (ii) whether antisense OPN expressing JB6 cells will suppress TPA-induced tumorigenic transformation and (iii) if suppressed TPA-induced cells transformation in the latter experiment is observed whether the addition of OPN will rescue this effect.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and reagents
Eagles Minimum Essential Medium (MEM) and phosphate-buffered saline were obtained from Mediatech, Herndon, UT. Fetal bovine serum (FBS) was obtained from Hyclone, Logan, UT. L-Glutamine, trypsin/EDTA, and antibiotics were purchased from Irvine, Santa Ana, CA. Hoechst 33258 and E-TOXATE® was purchased from Sigma (St Louis, MO). pTrcHisA and pEF/V5 vectors were purchased from InVitrogen (Carlsbad, CA). GRGDSP and GRGESP peptides were from Gibco BRL, Life Technologies (Grand Island, NY). Human fibronectin (FN) and antibody to rat FN were purchased from Boehringer Mannheim (Indianapolis, IN) and Chemicon (Temecula, CA), respectively.

Cell line
JB6 Cl41.5a mouse epidermal cells were generously provided by Dr N.Colburn from the National Cancer Institute (Frederick, MD). Cells were maintained in MEM supplemented with 5% heat inactivated FBS, 2% glutamine and 0.5% Fungi-Bact. The medium was changed every 2 days and cells were subcultured when they reached 80–90% confluence. To assess mycoplasma contamination, cells were checked routinely by DNA fluorochrome staining (61). Cell number was determined using a model ZM Coulter Counter.

Construction and purification of recombinant rOPN
The procedure to achieve recombinant rOPN expression has been described previously in detail (62). Briefly, mature rOPN expressed using the pTrcHisA vector has an additional 36 amino acids including six consecutive histidine residues at the N-terminus of the protein. The sequence and direction of the OPN cDNA insertion was confirmed by DNA sequencing. Based on SDS–PAGE analysis, the approximate purity of the protein was >95%. Amino acid sequencing at internal enterokinase cleavage sites, western blot analyses and mass spectrometry analysis (MALDI-TOF, LDE1002) confirmed that the isolated protein was rOPN. Using the Limulus Amebocyte lysate (E-TOXATE) to determine the endotoxin level of purified rOPN, we found that for 45 µg of rOPN, the endotoxin level was <1 ng.

Polyclonal antibody to rOPN
To generate a polyclonal antibody to recombinant rOPN, purified rOPN was run on a 10% SDS–polyacrylamide gel. The gel strips containing rOPN were sent to Research Genetics, Inc. (Huntsville, AL) for generation of a rabbit anti-rOPN antibody. The reactivity of rabbit antiserum to OPN was analyzed by western blot and found to specifically cross react with rOPN, mouse OPN (mOPN) and human OP (hOPN) of various forms (63). Antiserum to rOPN also immunoadsorbed phosphorylated and non-phosphorylated form of mOPN (see Figure 6). The goat anti-rat bone OPN antibody (generously provided by Dr Mary Farach-Carson) was used for Figure 1.



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Fig. 6. Synthesis and secretion of OPN by TPA-treated JB6 Cl41.5a cells, pEF-vector control (CT) clones and antisense OPN (AS) clones. (A) JB6 cells (lane 1) and AS clones 10, 13, 17, 25 (lanes 2–5, respectively) were treated with 16.2 nM TPA for 24 h and labeled with [35S]methionine for the last 3.5 h. Media collected were immunoadsorbed with antiserum to rOPN. Samples loaded onto the 10% SDS–PAGE were normalized to equal cell number (75,459 cells/lane). The fluorography is representative of one of duplicate samples. The film exposure time was 2 days. (B) JB6 cells (lanes 1 and 2) and CT clones 4 (lanes 3 and 4), 16 (lanes 5 and 6), 18 (lanes 7 and 8) and 3 (lanes 9 and 10) were treated with 0.001% DMSO (C) or 16.2 nM of TPA (T). Media collected were immunoadsorbed with antiserum to rOPN. Samples loaded onto the SDS–PAGE correspond to 63,706 cells/lane. Densitometric analysis indicated that the relative amount of TPA-induced secretion of OPN by clones CT 4, CT 16, CT 18 and CT 3 when compared with JB6 cells were 0.82, 0.77, 0.96 and 0.98, respectively.

 


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Fig. 1. Dexamethasone inhibits TPA-induced OPN mRNA and protein expression in JB6 cells. (A) Northern blot analysis of cells treated with 0.01% of DMSO (lane 1), 16.2 nM of TPA (lane 2), 250 nM of dexamethasone (lane 3) or both TPA + dexamethasone (lane 4) for 24 h. Graph is the average (n = 2) relative intensity of OPN steady-state mRNA normalized to ß-actin expressed in cells treated as described above. Dex, dexamethasone. (B) OPN protein expression of cells treated with DMSO (C, lane 1, right and left panels), dexamethasone (D, lane 2, right and left panels), TPA (T, lane 3, right and left panels) or both D and T (lane 4, right and left panels) with same concentration as above for 24 h. Cells were radiolabeled for the last 3.5 h as described in the Materials and methods. Left panel, immunoadsorbed secreted OPN labeled with [35]S methionine. The film exposure time was 52 h. Right panel, immunoadsorbed secreted OPN labeled with 32PO4. The film exposure time was 26 h. Samples loaded onto the SDS–PAGE correspond to 66,000 cells/lane for both gels.

 
Anchorage-independent transformation assay (soft-agar assay)
This procedure has been previously described (52). Briefly, 10,000 cells were suspended in 1.5 ml of 0.3% agar medium containing 10% FBS with or without matrix proteins or TPA and layered onto 7 ml of 0.5% solidified agar medium in 60 mm Petri dishes. In some instances, experiments were performed in 35 mm Petri dishes, where 3000 cells were mixed in 1 ml of 0.3% agar medium and poured on top of 4 ml of solid 0.5% agar medium. Each treatment was tested in triplicate dishes. The cells were incubated for 14 days at 37°C in a humidified atmosphere of 5% CO2 in air. Cell clusters with at least 10 cells were counted as a colony. The concentration of TPA and dexamethasone were 16.2 nM and 250 nM, respectively, for experiments in Figures 1 and 2. These drug concentrations were similar or less to experiments performed previously by others (60,64) for comparison purpose. For those anchorage-independent growth studies not involving dexamethasone treatment, 8.1 nM of TPA have been consistently used (65).



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Fig. 2. The inhibition of TPA-induced JB6 cell transformation by dexamethasone is partially rescued by the addition of OPN. Anchorage-independent growth assay of JB6 cells treated with 0.001% DMSO, 16.2 nM TPA (T), 250 nM dexamethasone (DEX), T + DEX or T + DEX + OPN (OP). Histogram represents two sets of experiments. Experiment 1 on the left side consists of JB6 cells incubated for 14 days with various drugs at both top and bottom agar layer except for those cells with T + DEX + OP no T + DEX was added to the bottom agar layer. Experiment 2 on the right side consists of JB6 cells incubated with various drugs at both top and bottom layer and with the addition of PBS to top layer corresponding to the same volume as the amount of OPN added. **P < 0.01 and *P < 0.05 significantly different from DMSO- or DMSO + PBS-treated cells. P < 0.05 significantly different from T + DEX- or T + DEX + PBS-treated cells. {circ}P < 0.05 significantly different from DEX-treated cells. Each bar represents the average of triplicate dishes with standard deviation.

 
Construction of antisense OPN expressing JB6 clones
The full-length mOPN cDNA, generously provided by Dr David Denhardt (Rutgers University, Piscataway, NJ) was inserted into the BamHI site of the pEF/V5 vector. The direction of insert was determined using restriction enzyme digestion and the integrity of the OPN cDNA was verified by sequencing. Purified endotoxin-free plasmids containing the antisense OPN cDNA or no insert (empty pEF/V5) were transfected into JB6 Cl41.5a cells using lipofectAMINE in MEM media. After 6 h, cells were changed to regular growth media. The next day, geneticin at 500 µg/ml was added to select for resistant clones. Between 45 and 48 geneticin-resistant clones containing anti-sense OPN cDNA (AS clones) or neo-vector (CT clones) were isolated starting after 10 days of incubation in selection media. These clones were expanded and frozen down prior to characterization.

Mycoplasma-free AS and CT clones were used for analyses. AS clones treated with TPA or DMSO for 24 h were screened for their production of OPN. Those AS clones, that suppressed TPA-induced OPN protein synthesis and secretion, as detected by metabolic labeling and immunoprecipitation (see below), were selected for soft agar assay. As for CT clones, those selected as negative controls had to pass the following criteria when compared with wild-type JB6 cells. It was necessary that they: (i) synthesize and secrete similar levels of OPN when treated with TPA and (ii) retain the ability to form colonies in soft agar in the presence of TPA and minimal colony formation when treated with DMSO as control.

Metabolic labeling and immunoadsorption of OPN
Procedures for metabolic radiolabeling and immunoadsorption of OPN have been established previously for JB6 cells (52,66). Briefly, JB6 cells were seeded at 20,000/cm2 in 24-well plates and grown to near confluence. Cell numbers were determined from parallel plates before and after 24 h of TPA or DMSO treatment (triplicate wells/treatment). Note, experiments involving cells grown in monolayer such as metabolic labeling, cell adhesion and northern blot analyses (Figures 3, 6 and 8), which has higher initial cell density than those of soft-agar assay (10,000 cells) were treated with 16.2 nM of TPA. This is consistent with the dose used in our previously published data (11,52,53,55,65). During the last 4 h of drug incubation, cells were incubated for 30 min in methionine- or phosphate-free medium containing the appropriate drug and then labeled with [35S]methionine or 32PO4 (50 µCi/ 0.5 ml medium) for the last 3.5 h. Media collected were immunoadsorbed as described previously (52,66) with antibody to recombinant rOPN or FN. Samples loaded onto the 10% SDS–polyacrylamide gel (SDS–PAGE) were normalized to equal cells number. Immunoadsorbed proteins were detected by treating the gels for fluorography and exposed to X-ray films.



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Fig. 3. rOPN promotes tumorigenic transformation of JB6 Cl41.5a cells. (A) 3000 cells were resuspended in 0.3% agar containing PBS or 5 (OP5), 15 (OP15) or 45 (OP45) µg/ml (0.11, 0.4, 1.2 mM, respectively) of rOPN and layered on top of 0.5% agar. The number colonies (containing 10 or more cells) were counted on day 14. *P < 0.05; **P < 0.01, significantly different from PBS-treated cells. {circ}P < 0.05, significantly different from OP45-treated cells. (A) (Inset) A full-length 10% SDS–polyacrylamide gel of purified histidine-tagged rOPN. Gel is stained with Coomassie Blue. Lanes 1 and 2 are 5 µg of rOPN from two different purification batches. The left column is low molecular weight standards. kD, kilodalton. (B) Different amounts of rOPN and 8.1 nM of TPA (T) were added to cells in 0.3% agar and layered on top of 0.5% agar. P < 0.01, significantly different from TPA-treated cells. *P < 0.05; **P < 0.01, significantly different from OP45 + T-treated cells. {circ}{circ}P < 0.01, significantly different from DMSO-treated cells.

 


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Fig. 8. TPA stimulates FN synthesis and secretion and cell proliferation in JB6 Cl41.5a cells and AS clones. (A) Cells were treated with 16.2 nM of TPA or DMSO 0.0001% for 24 h and labeled with [35S]methionine for the last 3.5 h. Media collected were immunoadsorbed with antibody to FN. For DMSO-treated samples (left panel), the volumes loaded onto the SDS–PAGE correspond to 59,209 cells/lane. Lane 1, JB6 cells, lanes 2–6 are clones CT18, CT3, AS13, AS17 and AS25. Film exposure time to fluorogram was 1 week. For TPA-treated samples (right panel), the volumes loaded onto the SDS–PAGE correspond to 93,078 cells/lane. Lane 1, JB6 cells; lanes 2–6 are clones CT3, CT18, AS13, AS17 and AS25, respectively. Film exposure time to fluorogram was 2 days. (B) Cells were seed at 30,000 cell/cm2 in 24 well plates in triplicate wells/treatment. Media were changed on days 0, 2 and 3 containing 0.001% DMSO (C) or 16.2 nM of TPA (T). Cells were counted on days 0 and 4. *P < 0.05 significantly different from cells treated with DMSO.

 
RNA isolation and northern blot analyses
Total cellular RNA was isolated from JB6 cells using a previously described procedure (55) with slight modification. Ten micrograms of total RNA was denatured and loaded onto 1% agarose formaldehyde-containing gels. Gels were run in the absence of ethidium bromide at 60 V for 2.5 h. RNA was transferred onto Sure BlotTM positively charged nylon membranes (Oncor, Gaitherburg, MD) by capillary action overnight and immobilized on the membrane by incubating 30 min at 80°C. Blots were stained with methylene blue to determine efficiency of transfer and to ascertain RNA integrity based on the prominence of 18S and 28S ribosomal RNA bands.

Northern blots were performed as described previously (55). Briefly, cDNA probes (mOPN; a gift from Dr David Denhardt; Rutgers University, Piscataway, NJ), or human ß-actin were labeled with [{alpha}-32P]dCTP using random priming according to manufacturer's instructions (Stratagene, La Jolla, CA). Hybridization of the probe to the mRNA was allowed to proceed overnight, and membranes were washed and exposed to film (Scientific Imaging film XAR; Eastman Kodak, Rochester, NY) at -20°C then developed. Autoradiographs of Northern blots generated as described above were used in densitometric scanning (Multi-Analyst; Bio-Rad Laboratories, Inc., Hercules, CA) to obtain relative units of OPN mRNA normalized to levels of ß-actin mRNA.

Statistics
Statistical analyses were performed using Student's unpaired t test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Dexamethasone inhibits TPA-induced OPN mRNA and its protein expression in JB6 Cl41.5a cells
To determine whether induced OPN is an important player downstream of tumor promoter-induced transformation in JB6 cells, we postulated that tumor promoter inhibitors such as glucocorticoid (5659), should not only suppress tumor promoter induced transformation and concomitantly attenuate OPN expression, as shown previously (44,60), but that the addition of OPN should rescue the suppressed transformation of JB6 Cl41.5a cells. Although previous studies using JB6 Cl22 have shown that 1 µM of dexamethasone or fluocinolone acetonide (both synthetic analogs of glucocorticoid) suppressed TPA-induced OPN mRNA (60) and that 100 nM of fluocinolone acetonide (44), inhibited TPA-induced tumorigenic transformation of JB6 Cl41, it was necessary to also determine whether the TPA-treated JB6 Cl41.5a cells (a subclone of JB6 Cl41) responded similarly to dexamethasone.

We first determined the effect of dexamethasone on TPA-induced OPN synthesis and secretion in JB6 Cl41.5a cells. Treatment of JB6 cells with dexamethasone alone for 24 h did not induce OPN mRNA (Figure 1A, top panel, lanes 3). Instead, dexamethasone markedly inhibited TPA-induced OPN mRNA by 60% (Figure 1A, top panel 4, see relative expression in lower panel), which subsequently resulted in the decreased synthesis and secretion of the total OPN protein by 80% (Figure 1B, [35S]methionine-labeled OPN, left panel, lane 4) and the phosphorylated form of OPN by 50% (Figure 1B, 32PO4-labeled OPN, right panel, lane 4). Our results showed that dexamethasone also suppressed TPA-induced OPN mRNA expression in JB6 Cl41.5a cells as shown previously in another promotable JB6 Cl22 cells (60).

Dexamethasone suppresses TPA-induced tumorigenic transformation of JB6 Cl41.5a cells and the addition of OPN partially rescues this effect
Concomitant to inhibiting TPA-induced OPN mRNA expression, dexamethasone also suppressed TPA-induced tumorigenic transformation of JB6 Cl41.5a cells by 94 and 93% in experiments 1 and 2, respectively (Figure 2). Dexamethasone alone did not induce anchorage-independent colony formation. Our findings are consistent with studies using fluocinolone acetonide in TPA-treated JB6 Cl41 cells (44).

To determine whether the suppression of OPN might be an independent effect of the suppression activity of dexamethasone on tumorigenesis, we added exogenous OPN to TPA and dexamethasone-treated cells to observe whether OPN could specifically rescue the observed suppression by dexamethasone. Figure 2, experiment 1 showed that the addition of OPN rescued these cells from dexamethasone suppression of TPA-induced colony formation in soft agar. We also found that the lack of addition of TPA and dexamethasone to the lower 0.5% agar layer in 60 mm dishes containing exogenous OPN (Figure 2, Exp 1) resulted in a greater capacity of OPN to reverse dexamethasone inhibition of TPA-induced anchorage-independent growth versus those cells treated with drugs in both agar layers (Figure 2, Exp 2). Collectively, the data suggest that the reduction by dexamethasone on TPA-induced cell transformation is associated partially with the decrease in OPN expression.

OPN dose-dependently promotes tumorigenic transformation of JB6 cells
Although, the data shown above suggest that TPA-induced OPN may play a critical role in tumorigenic transformation of the promotable JB6 cells, to more specifically test the requirement of OPN we determine if OPN overexpression (addition of excess OPN) promotes tumorigenic transformation of JB6 Cl41.5a cells. Thus, anchorage-independent growth assays were performed using purified histidine-tagged rOPN added to the cells (Figure 3A, inset). The addition of increasing amounts of exogenous rOPN (5–45 µg/ml) to JB6 cells showed a significant dose-dependent increase in the number of transformed colonies compared with control PBS-treated cells (Figure 3A). Incubation of JB6 cells with 45 µg/ml of rOPN resulted in 3.4-fold higher number of colony formation compared with control cells. Furthermore, an additive increase in the number of colonies was observed when increasing doses of rOPN were added to TPA-treated cells (Figure 3B). Thus, OPN alone promotes JB6 cell transformation.

Neither FN nor BSA promotes tumorigenic transformation of JB6 cells
Although, JB6 cells normally synthesize minimal amounts of OPN until they are treated with the tumor promoter, TPA (50,52,53), the adhesive protein FN is constitutively synthesized and TPA also enhances FN synthesis and secretion (67) (see also Figure 8). Therefore, to determine whether the elevated expression of FN induced by TPA might also stimulate transformation of JB6 cells, anchorage-independent growth assay was performed by incubating JB6 cells with 45 µg/ml of human FN, rOPN or BSA as negative control. Neither FN- nor BSA-treated JB6 cells showed enhanced cell transformation or colony formation over that of control cells, while rOPN at the same concentration markedly enhanced (P < 0.05) cell transformation (Figure 4).



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Fig. 4. FN does not induce cell transformation of JB6 cells. Cells were resuspended in 0.3% agar containing PBS or 45 µg/ml of rOPN, FN or BSA. *P < 0.05, significantly different from PBS-, BSA- and FN-treated cells.

 
Purified antibody to OPN attenuates OPN induced cells transformation, but RGD peptides fail to do so
To determine whether OPN enhancement of anchorage-independent growth is mediated through its RGD-cell-binding sequences. Cells were premixed with the competitive RGD or the control RGE (argininylglycylglutamic acid) peptides at 1 or 10 µM prior to seeding on top of the 0.5% agar. RGD peptides failed to inhibit OPN from stimulating colony formation (Figure 5A), suggesting that the observed OPN effect on transformation may not be mediated through its RGD motif. However, pre-incubation of purified polyclonal antibody to OPN or IgG with molar equivalence of 45 µg/ml of OPN for 30 min significantly inhibited OPN from promoting anchorage-independent growth of JB6 cells (Figure 5B).



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Fig. 5. RGD peptides do not inhibit OPN-induced JB6 cell transformation, however, antibody to OPN significantly reduces OPN-induced colony formation. (A) Cells were pre-incubated with GRGDSP (competitive) or GRGESP (negative control) peptides at 1 or 10 mM for 20 min prior to seeding on top of the 0.5% solidified agar layer with or without 45 µg/ml of OPN and incubated for 14 days. **P < 0.01 significantly different from respective controls, which do not have OPN (designated as OP or P) added. (B) OPN were pre-incubated with molar equivalent of purified anti-OPN or control IgG for 30 min prior to mixing with cells and layered on top of the 0.5% solidified agar. Negative control such as cells incubated with PBS, anti-OPN or IgG were also included. Histogram was plotted as the percent of colony formation relative to OPN-treated cells. The number of clones for the negative controls was in the range of 60 to 35 clones/10,000 cells. *P < 0.05 significantly different OPN (OP)-treated cells.

 
Stably transfected antisense OPN clones show attenuation in TPA-induced OPN synthesis and secretion
Additionally, we determined whether suppressing the endogenous synthesis of TPA-induced OPN, by antisense OPN expression, would result in a reduction of TPA-induced JB6 cell transformation. Thus, stably transfected JB6 clones containing pEF vector carrying the antisense OPN cDNA (AS) driven by human elongation factor 1{alpha}-subunit promoter were established and characterized for TPA-induced OPN synthesis and secretion prior to performing the cell transformation experiments. Forty-eight geneticin-resistant clones were isolated, expanded and characterized for their ability to synthesize and secrete OPN. Clones were seeded at equal density, treated with TPA at near confluent state and radiolabeled with [35S]methionine. Medium from each individual clone was immunoadsorbed with antibody to OPN and loaded according to equal cell number onto SDS–PAGE (52,66). Out of all 48 clones characterized, there were only four clones that showed significant reduction in TPA-induced OPN synthesis and secretion. Figure 6A shows that compared with JB6 wild-type cells (lane 1) AS clones 10, 13, 17 and 25 (lanes 2–5) markedly exhibit reduced TPA-induced OPN expression by 33, 45, 67 and 58%, respectively. From an average of two repeated experiments the percent reduction of TPA-induced OPN secretion by AS clones 10, 13, 17 and 25 compared with that of JB6 wild-type cells were 38 ± 8, 46 ± 2, 59 ± 14 and 56 ± 3, respectively.

Characterization and selection of control clones stably transfected with pEF vector
Clones stably transfected with pEF vector only or neo-vector control (CT) were also characterized. Out of forty-five clones isolated, 31 individual CT clones were screened to select for those that have similar ability as the wild-type JB6 cells to form colonies in soft agar in the presence of TPA. We found that compared with TPA-treated JB6 cells, nine clones (29%) had lower TPA-induced colony formation, four clones (13%) showed higher TPA-induced colony formation and 18 clones (58%) showed similar levels of colony formation. From the latter group, we selected four clones, which also have low colony formation when treated with 0.001% DMSO used as negative control.

Those CT clones, which showed similar TPA-induced anchorage-independent growth as the wild-type JB6 cells, were then tested for their ability to synthesize and secrete similar levels of TPA-induced OPN. Densitometric analysis of bands in Figure 6B indicated that CT18 and CT3 clones have similar levels of TPA-induced OPN secretion as the wild-type JB6 cells, while CT4 and CT16 clones showed slight reduction in OPN secretion, thus CT3 and CT18 were used as our positive controls in the following experiments.

Antisense OPN expressing clones attenuate TPA-induced tumorigenic transformation of JB6 cells and the addition of OPN partially reverses this effect
To determine whether the AS clones with reduced TPA-induced OPN expression also suppressed TPA-induced tumorigenic transformation of JB6 cells anchorage-independent growth assays were performed using JB6 cells, vector only clones (CT3 and CT18) and antisense OPN clones (AS10, AS13, AS17 and AS25). TPA-treated CT clones containing neo vectors showed slightly lower colony formation than those of wild-type JB6 cells treated with TPA (Figure 7). Comparison of TPA-treated CT3 and CT18 clones with those of TPA-treated AS clones showed that AS clones had significantly lower ability to induce colony formation on soft agar. The percent reduction of cell transformed in TPA-treated clones AS10, 13, 17 and 25 cells relative to the average of CT3 and CT18 cells were 46, 86, 95 and 77%, respectively. Comparison of the relative OPN expression levels in the TPA-treated antisense clones (Figure 6A) with the relative inhibition of transformation (Figure 7) indicated that the lowest OPN expressor (AS17 clone) is the most inhibited for transformation response, the highest OPN expressor (AS10 clone) is the least inhibited for transformation and the intermediate expressors showed intermediate inhibition of transformation. This trend suggests that OPN concentration may be limiting for transformation response.



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Fig. 7. Anchorage-independent growth assay of TPA-treated JB6 cells, CT and AS clones with or without OPN. JB6 cells and vector only clones CT3 and CT18 were treated with 0.001% DMSO or 8.1 nM TPA. Antisense clones AS10, AS13, AS17 and AS25 were treated with 0.001% DMSO or 8.1 nM TPA with or without 45 µg/ml OPN. **P < 0.01 and *P < 0.05 significantly different from DMSO-treated cells. ••P < 0.01 and P < 0.05 significantly different from TPA-treated cells. {circ}P < 0.05 significantly different from TPA-treated JB6 cells. Each bar represents the average of triplicate dishes with standard deviation. Data represent one of two repeated experiments.

 
Moreover, to test whether these AS clones with suppressed OPN secretion could restore their ability to grow anchorage-independently we added exogenous OPN. Figure 7 showed that the addition of OPN to AS clones completely rescued AS10 cells' ability to grow anchorage-independently in the presence of TPA, while partially rescued other AS clones from inhibition of tumorigenic transformation. The data provide additional evidence that OPN has a functional role in tumorigenesis of these cells.

Antisense OPN and control clones are responsive to TPA-induced FN production and cell proliferation
To test the possibility that the observed reduction of TPA-induced anchorage-independent growth of AS clones might be due to other changes such as the lack of TPA responsiveness rather than to specific reduction of OPN production, we determined whether TPA still induces FN synthesis and secretion and cell proliferation in a monolayer system. Metabolic labeling with [35S]methionine of TPA-treated (Figure 8A, right panel) or non-treated (Figure 8A, left panel) CT and AS clones followed by immunoadsorption of these conditioned media with anti-FN antibody indicated that the synthesis of FN was induced by TPA in AS clones to similar levels as those of CT clones and JB6 wild-type cells (Figure 8A, right panel).

Additionally, when AS clones were grown in monolayer and treated with TPA for 4 days, these cells, like wild-type JB6 cells, were still responsive to the mitogenic effect of TPA (Figure 8B). Thus, AS clones' ability to inhibit TPA-induced cell transformation is specific to the suppressed level of TPA-induced OPN synthesis and not due to AS clones' unresponsiveness to TPA.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The application of the non-malignant JB6 cells, which lacks both the mutated p53 and ras genes (43), is a useful model to study the requirement of OPN induction in traversing the rate-limiting step, tumor promotion. Furthermore, TPA-induced OPN expression has been postulated to involve the activation of AP-1 (68) mediated upstream by novel isoforms of PKCs, MEK and MAPK (55). Since activation of AP-1 by TPA has been shown to be a required event in both JB6 model and in vivo tumor promotion studies (44,45), it is conceivable that the rapid and persistent induction of OPN expression and secretion downstream of AP-1 is required for tumor promotion.

Additionally, synthetic analogs of glucocorticoid, known tumor promoter inhibitor in vivo, have been shown to suppress TPA-induced AP-1 transactivation and cell transformation in JB6 Cl41 (44) and inhibit TPA-induced OPN expression in JB6 Cl22 cells (60). We have confirmed previous studies using dexamethasone on another promotable JB6 Cl41.5a cell suggesting that the observed effect in other JB6 clones was not merely a clonal effect. Furthermore, we have also shown that the suppression of OPN is not independent of the effect of dexamethasone suppression on tumorigenesis, since the addition of OPN partially rescued the suppression of TPA-induced colony formation by dexamethasone. The ability of OPN to reverse this suppression was inversely dependent on the amount of dexamethasone present in the agar. The data suggest that OPN induction is sufficient to induce transformation of JB6 cells. To further support the importance of OPN in tumor promotion, retinoic acid, another tumor promoter inhibitor in vivo (69,70), has also been shown to suppress TPA-induced OPN expression (60) and cell transformation of the promotable JB6 cells (44).

More specifically, direct study on the requirement of OPN in transformation of JB6 Cl41.5a cells showed that the addition of high concentration of rOPN (45 µg/ml) promotes ~3–10% of JB6 cells to transform even without the addition of TPA, confirming that OPN induction alone is sufficient to transform these cells. In contrast to rOPN, exogenous addition of FN, which is an adhesive protein constitutively secreted by JB6 cells, did not induce cell transformation. Additionally, it is unlikely that vitronectin (VN), another RGD-containing matrix protein not produced by JB6 cells (11), but found in FBS, would have the capacity to stimulate JB6 cell transformation. VN and also FN are both found in 10% FBS used in the soft agar assay in concentrations of 20 µg/ml and 15–18 µg/ml, respectively (ref. 71 and personal communication with Hyclone). Comparison of the total moles of FN and VN contributed by FBS in agar per dish to that of the added rOPN showed that the amounts were comparable. Since minimal transforming ability was observed in JB6 cells treated with PBS only (negative control), this implies that both FN and VN do not have the ability to induce tumorigenic transformation of JB6 cells.

Although rOPN alone can promote the anchorage-independent growth of JB6 cells, the percent of cells transformed by rOPN is significantly less than the percent of cells transformed by the tumor promoter, TPA. The difference may be due to the fact that the addition of exogenous OPN to cell suspension in 0.3% agar immediately prior to agar solidification will not allow all the added OPN to be in direct contact with the cells. Whereas, the treatment of TPA on JB6 cells not only stimulates the production and secretion of OPN, but that it also enhances cell adhesion to OPN (11). Therefore, the addition of TPA in an anchorage-independent growth assay should increase secreted OPN localized to the surface of the cells, which are embedded in soft agar, and perhaps also increase the cell surface receptors' affinity to OPN and consequently, facilitate OPN induced cell transformation by an autocrine pathway. This reasoning is further supported by the observation that concurrent treatment with rOPN and TPA resulted in an additive effect in transforming JB6 cells. Besides OPN, FN is another matrix, adhesive protein stimulated by TPA. However, it is unlikely that the additive effect of cell transformation is due to FN. Previous studies have shown that TPA-treatment decreases cell surface association of JB6 cells for FN (11,67) possibly by reducing the cell surface expression of the integrin ß1 subunit, which can result in decreasing the surface expression of the FN integrin {alpha}5ß1 (11).

Purified polyclonal antibody specific to OPN at molar equivalent significantly inhibited OPN-induced cell transformation indicating specificity. However, the addition of competitive RGD peptides did not suppress OPN-induced tumorigenic transformation of JB6 cells suggesting that integrin receptors, such as {alpha}vß5, reported to most likely mediate TPA-treated JB6 cell adhesion to OPN coated plates (11), may not be involved. It is possible that TPA-induced OPN in suspension/soft agar assay stimulates JB6 cell transformation through a non-RGD region by interacting with other integrins (31,33,72) or the hyaluronic acid receptor CD 44 (34,35), which is also expressed in JB6 cells (11). Further studies will be necessary to determine the specific receptor involved in OPN induction of JB6 cell transformation.

Additional evidence supporting the requirement of OPN induction in tumor promotion is the finding that stably transfected antisense-OPN AS clone with suppressed TPA-induced OPN synthesis and secretion showed decreased TPA-induced cell transformation. We believe that the decrease in TPA-induced cell transformation is due to the suppressed expression of OPN and not due to the non-responsiveness to TPA since FN synthesis and cell proliferation in these anti-sense OPN clones were both induced by TPA. Furthermore, the addition of exogenous OPN significantly reversed the suppressed TPA-induced cell transformation of AS clones. Although, only clusters of at least 10 or more cells were counted as transformed clones in all anchorage-independent growth assays, we noticed in the latter experiment that a significant number of clusters with four to eight cells were present in TPA-treated AS clones containing OPN compared with those without OPN. Collectively, these observations confirm the requirement of OPN induction in TPA-induced colony formation.

Although, previous attempts to determine whether OPN induction is required has been performed in JB6 cells (64), the findings were inconclusive due to the use of dexamethasone as the inducer of antisense OPN expression. Dexamethasone, as others and we have shown, suppressed TPA-induced OPN expression and tumorigenic transformation of JB6 cells.

It is conceivable that aberrant/over-expression of OPN in vivo can induce or promote tumor formation as recent generation of homozygous transgenic OPN mice showed that high mortality rate of these mice resulted from spontaneous malignant tumors in various organs, which normally do not constitutively express high levels of OPN (73). We hypothesize that the ability of OPN to induce tumorigenic transformation of JB6 cells is through either prevention of anoikis (suspension-induced apoptosis) and/or stimulating cell proliferation. Several studies in other cell systems have shown that OPN has the ability to prevent apoptosis and promote cell proliferation (5,74). OPN has also been reported to adhere to cell surface integrin such as {alpha}vß3 or CD44 and subsequently bind to complement factor H resulting in the evasion of complement-mediated cell lysis (75). Thus, OPN in vivo may assist in enhancing the survival of initiated or chromosomally altered cells through evading cell lysis and promote their proliferation, which subsequently result in clonal expansion or tumor formation.

In summary, we have conclusively showed that OPN induction is required and can be sufficient to induce transformation in the mouse JB6 model. Studies are in progress to determine the mechanism by which OPN facilitates tumorigenic transformation of JB6 cells. To complement our in vitro experiments, mouse models are being developed to study the requirement of OPN induction in tumor promotion in vivo.


    Notes
 
4 To whom correspondence should be addressed Email: plchang{at}uab.edu Back


    Acknowledgments
 
We thank Xiuxiang Jiao for her technical assistance and Drs Craig Elmets, Richard Mayne and Wun-Ling Chang for their critical review of the manuscript. We also thank Lori Coward for performing the MALDI-TOF on purified rOPN. This work was supported by R29 CA69688 and R01 CA90920 to P.-L.Chang and by R25 CA76023–02 ‘UAB Cancer Research Experiences for Students’ (CaRES) summer program 1999 to M.Cao.


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 Materials and methods
 Results
 Discussion
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Received May 12, 2003; revised July 16, 2003; accepted July 29, 2003.