The immortalized non-tumorigenic mouse fibroblast NIH3T3 cell line was obtained from the American Type Culture Collection (Rockville, MD). NIH3T3 cells were grown in DMEM (Wisent, St-Bruno, QC, Canada) supplemented with 10% FBS (Wisent), 2 mM L-glutamine (Wisent), 100 U/ml penicillin and 100 μg/ml streptomycin. 293T cells were cultured in DMEM (Wisent) supplemented with 10% FBS, 0.1 mM MEM non-essential amino acid solution and 10 mM HEPES. Cells were maintained at 37°C in humidified 5% CO₂ incubators. MUC16 C-terminal domain (MUC16 CTD) (containing last 284 C-terminal residues) was amplified by PCR from pME18S-FLJ14303 (Helix Research Institute, Japan). The 5′ primer integrated an EcoRV restriction site while the 3′ primer integrated a XhoI restriction site. The digested amplicon was inserted in a pLenti6V5 vector containing at the 5′ end an IgK leader sequence, a myc-tag and a small linker (23 residues) and at the 3′ end a linker sequence and a Flag-tag (21 residues) (33,34). The control vector lacked the amplified MUC16 CTD sequence. MUC16 TMU contains the extracellular unique region and the transmembrane domain. To ensure anchorage of the construction at the cytoplasmic membrane, three residues adjacent to the transmembrane domain in the cytoplasmic tail were conserved in MUC16 TMU. After verification by sequencing, 293T cells were transfected with equal amout of pLenti vectors Plp1, Plp2, PlpVSVG and either the pLenti6V5-MUC16-CTD, -EV or -MUC16 TMU using Lipofectamine 2000 (Life Technologies, Burlington, ON, Canada). Lentivirus produced by 293T cells were collected 48-h post-transfection and kept at −80°C. NIH3T3 cells were plated in 6-well plates at 90% confluence in complete medium DMEM. Polybrene (4 μg/ml final concentration) was added to the vial of thaw lentiviruses. This mixture was incubated with the cells for 1 h at 37°C, 5% CO₂. After the initial incubation with the viruses, complete medium supplemented with polybrene (without antibiotics) was added. Forty-eight hours later the culture media was replaced with complete media containing blasticidin (0.5 μg/ml) (Sigma Canada, Ltd., Oakville, ON, Canada). NIH3T3 expressing either empty vector, MUC16 TMU or MUC16 CTD populations were obtained while maintaining the blasticidin selection throughout. Validation of expression for the populations was done by RT-PCR and WB analysis. Independent clones for MUC16 CTD-expressing NIH3T3 cells were obtained by limiting dilution in a 96-well plate with blasticidin selection.

Primers for PCR amplification of MUC16 CTD were as follow: 5′-CAACTCGAGTTGCAGATCCTCCAGGTC TAG-3′ (sense) and 5′-AACGATATCACCCTGCTGAGGGAC ATC-3′ (antisense). Primers for PCR amplification of MUC16 TMU were as follow: 5′-AATATCTTAAGCGGCCGCACCG GGTGGTCACCAGGAC-3′ (sense) and 5′-ATGCGGCCAT CTAGGCCACATCACCCTGAGGGAC-3′ (antisense) (Integrated DNA Technologies, Inc., Coralville, IA). PCR was performed as follows: 94°C for 5 min followed by 30 cycles of 94°C for 1 min, 65°C for 1 min and 72°C for 1 min and finalized at 72°C for 15 min.

Cell were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.1% SDS, 150 mM NaCl, 0.5 mM EGTA, 1% Triton X-100) with freshly added protease inhibitors [1 μg/ml 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 2 μg/ml aprotinin, 0.7 μg/ml pepstatin and 0.5 μg/ml leupeptin] (Sigma) and proteins were quantified by Bradford assay (Bio-Rad, Mississauga, ON, Canada).

Cells lysates (equal amounts of proteins) were submitted to SDS-PAGE electrophoresis (10%) and transferred onto PVDF membrane (Roche, Laval, QC). The membranes were blocked with 5% non-fat milk and probed with indicated antibodies. Anti-Flag M2 antibody (1:500) and anti-α-tubulin (1:25000) antibodies were purchased from Sigma. Anti-c-myc sc-789 antibody (1:1000) and anti-ERK 1: sc-93 (1:250) were from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-phospho-p44/42 MAPK (1:1000), anti-AKT (1:1000), anti-Bcl-x_L (1:1000), anti-cyclin A2 (1:1000), anti-cyclin D1 (1:1000), anti-cyclin D2 (1:1000), anti-cyclin E2 (1:1000) and HRP-conjugated rabbit anti-mouse antibody and anti-rabbit antibody were all from Cell Signaling Technology (Danvers, MA, USA). Anti-Mcl1 (1:500) was purchased from BD Bioscience (Mississauga, ON, Canada). Anti-Bcl-2 (1:1000) antibody was from Dako (Burlington, ON, Canada). Anti-phospho-Akt (Ser 473) was from Life Technologies. The immunoblots were developed with chemiluminescence using the ECL system according to the manufacturer’s instruction (GE Healthcare, Baie d’Urfe, QC).

Cells (1×10⁵) were suspended in complete medium containing 0.33% of melted bacto-agar. The resulting suspension was added to a 60-mm plate covered with a 2-ml layer of solidified 0.66% bacto-agar in complete medium. Grossly visible colonies were stained with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma) and counted after 30 days of growth at 37°C in 5% CO₂ incubators. Three experiments were performed in triplicate. The mean ± SD was calculated from three independent experiments.

NIH3T3 clones were grown on glass slides until a 50–70% confluence was reached. Glass slides were then washed in cold PBS and cells fixed in 3.7% formaldehyde (Fisher) for 10 min. Glass slides were next rinsed for 5 min in PBS and quenched (glycin 0.1 M) during 30 min. Depending on the experiment, cells were permeabilized in PBS containing 0.2% Triton X-100 for 20 min at room temperature. Slides were rinsed twice in PBS and blocked overnight in PBS/2% goat serum at 4°C. Slides were washed 3 times in PBS, incubated with primary anti-c-myc sc-789 (1:200) antibodies in blocking buffer at room temperature for 1 h. Slides were washed 3 times in PBS, incubated for 45 min at room temperature with Alexa FluoR 594 or F(ab’)2 fragment of goat anti-mouse or goat anti-rabbit IgG from Life Technologies. After washing, slides were incubated for 2 min in 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei, washed again in PBS and mounted for visualization by fluorescence microscopy with an Olympus IX70 (Olympus, Hamburg, Germany). Expression of MUC16 CTD was detected using anti-c-myc sc-789 antibody (1:200).

Cell growth was determined by XTT assay. Briefly, cells were resuspended in complete medium and plated into 96-well plates at a density of 1×10⁴ cells/well. Cells were cultured for various times in complete and low serum concentration before adding XTT reagents. After incubating for 1 h at room temperature, the absorbance was read at 450 nm using a microplate reader at 450 nm (Tecan Sunrise, Research Triangle Park, NC, USA). All assays were performed in triplicate and mean ± SEM was calculated from three independent experiments.

Cells were seeded in 6-well plates in complete medium. Fourteen days post-confluence, the cells were fixed and stained with crystal violet (Sigma) and the number of colonies composed of >50 cells was determined. All assays were performed three times. The mean ± SEM was calculated from three independent experiments.

A drop of 15 μl medium containing 5×10³ cells was pipetted onto the inner surface of a Petri dish lid. The lid was then placed on the Petri dish so that the drops were hanging from the lid with the cells suspended within them. To eliminate evaporation, 10 ml PBS was placed in the bottom of the Petri dish. During 5 days of incubation at 37°C in 5% CO₂ incubators, the lid of the Petri dish was inverted and photographed using a microscope at ×100 magnification.

Mice (athymic nu/nu) were purchased from Charles River (St-Constant, QC, Canada) and housed in pathogen-free animal facilities. Mice were treated according to the Institution Animal Care guidelines. To assess tumorigenic capacity of EV-, MUC16 CTD- and MUC16 TMU-expressing NIH3T3 cells, 5×10⁶ cells were suspended in serum-free medium and injected subcutaneously into 6–8 weeks old female athymic CD-1 nude mice. Each mouse received 4 injections in the flanks and each group contained 2 mice. Tumors were excised and weighted after 23 days. The mean tumor weight ±SEM was calculated from eight independent site injections.

Statistical analysis was done using the BMDP statistical analysis software (UCLA, Los Angeles, CA). The threshold for statistical significance is a probability of 0.05. In the nude mouse model, data were expressed as mean ± SEM of tumor weight. The two-sided Student’s t-test was used to assess statistical differences between groups for the in vivo mouse models.

To assess the potential of MUC16 in mediating oncogenic transformation, we infected the MUC16 negative immortalized non-tumorigenic NIH3T3 mouse fibroblast cell line with lentiviruses to generate NIH3T3 cell lines expressing empty vector (EV), MUC16 TMU or MUC16 CTD. MUC16 CTD consists of the last C-terminal 283 residues which include the transmembrane domain (TM) and cytoplasmic tail (CT) (Fig. 1A). Because of the lack of available antibody against MUC16 CTD, the construct was tagged with c-myc epitope at the N-terminus and a Flag epitope at the C-terminus (Fig. 1A). MUC16 TMU is similar to MUC16 CTD except that the CT domain was deleted (Fig. 1A). Stable clones were screened for MUC16 CTD expression and two independent clones that exhibited MUC16 CTD expression were selected (CTD1 and CTD2). Empty vector and MUC16 TMU consisted of pooled populations. The expression of MUC16 CTD in NIH3T3 cells was validated by RT-PCR (Fig. 1B) and immunoblotting with anti-c-myc and anti-Flag antibodies (Fig. 1C). The expression of MUC16 CTD and MUC16 TMU was further characterized by immunofluorescence with anti-c-myc antibody on non-permeabilized cells (Fig. 1D).

One of the hallmarks of transformed cells is their ability to grow in the absence of exogenous factors, which is shown by the ability to grow in low serum conditions (35). To assess the effects of MUC16 expression on cell growth, EV-, MUC16 TMU and MUC16 CTD-expressing NIH3T3 cells were seeded at low density and the number of viable cells was assessed at different time intervals for up to 6 days in normal (10%) and low (1%) serum concentration (Fig. 2A). Expression of MUC16 CTD in both MUC16 CTD1 and MUC16 CTD2 led to significantly accelerated cell growth under both normal (10%) and low (1%) serum conditions when compared to cells expressing the empty vector or MUC16 TMU.

To further determine growth-related features, we assessed the capacity of cells to form three-dimensional structures using the hanging droplets technique (36). Larger spheroids were observed for MUC16 CTD-expressing NIH3T3 at Days 1 and 5 when compared to EV-expressing cells (Fig. 2B and C). EV-expressing NIH3T3 cells were able to form very compact spheroids whereas spheroids formed by MUC16 CTD-expressing cells were not as compact with cells detaching from the spheroid.

The effects of MUC16 expression on colony formation was assessed under anchorage-dependent and anchorage-independent conditions. Cells were seeded at low density and after 2 weeks (post-confluence), they were fixed and stained with crystal violet (Fig. 3A). MUC16 CTD-expressing NIH3T3 cells were characterized by loss of contact inhibition, overlapping growth and showed significantly (P<0.001) enhanced foci formation when compared to MUC16 TMU-transfected cells. Anchorage-independent growth was assessed by the formation of colony in soft agar. Under these conditions, there were few colonies with NIH3T3 cells expressing the empty vector or MUC16 TMU (Fig. 3B). NIH3T3 cells have been reported to occasionally spontaneously transform (37). More importantly, the number of soft agar colonies was markedly (P<0.001) increased with NIH3T3 cells expressing MUC16 CTD when compared to MUC16 TMU or EV-expressing cells. These data suggest that MUC16 CTD expression induces the transformation of NIH3T3 cells.

To further assess the oncogenic potential of MUC16 CTD expression, we examine the formation of subcutaneous tumors in mice injected with MUC16 CTD and MUC16 TMU. Because MUC16 CTD1 and MUC16 CTD2 expression resulted in similar phenotype in vitro, only MUC16 CTD1-expressing cells were further selected for in vivo experiments. Mice injected with MUC16 CTD1-expressing NIH3T3 cells grew much larger tumors over time when compared to MUC16 TMU-expressing cells (Fig. 4A and B). The average tumor volume in NIH3T3 MUC16 CTD1 cell-injected mice exceeded 0.25 g at 23 days. In contrast, tumors from NIH3T3 MUC16 TMU-injected mice were 0.067 g at 23 days. The expression of MUC16 CTD and MUC16 TMU in the subcutaneous tumors was confirmed by RT-PCR (Fig. 4C).

MUC16 CTD expression increases NIH3T3 cell proliferation in vitro (Fig. 2) and in vivo (Fig. 4). We used immunoblot analysis to investigate the association between MUC16 CTD expression and the corresponding changes in the expression of proteins that are involved in cell cycle regulation, including ERK1/2, Akt, cyclin D1, cyclin D2, cyclin E and cyclin A. NIH3T3 cells expressing MUC16 CTD showed increase phosphorylation of the cell cycle regulatory protein ERK1/2 and had higher levels of G1-S cell cycle transition regulatory cyclin D2 and the short form of S-phase regulatory protein cyclin E that usually increases cell proliferation (Fig. 5). The presence of the shorter form of cyclin E is usually found in tumors of ovarian cancer cells, but not in normal cells (38). The levels of G2-M phase regulatory protein cyclin A1 was decreased whereas cyclin B1, another G2-M phase regulatory protein, was unchanged. The expression of proteins usually associated with cell survival such as Akt, Mcl-1, Bcl-2 and Bcl-X_L were unaffected by MUC16 CTD expression. Thus, MUC16 CTD expression is associated with increased expression of cell cycle regulatory proteins.