Antibodies against E2F4 (C-20), p130 (C-20), DP-2 (C-20), and HA-probe (F-7) were all purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). β-actin monoclonal antibody (clone C-4) was obtained from Millipore (Billerica, MA, USA). Cycloheximide was purchased from Calbiochem (San Diego, CA, USA). All other materials were from Sigma-Aldrich (Oakville, ON, Canada) unless stated otherwise.

The expression vectors (pCDNAneo3) encoding for E2F4 and p130 were obtained from Dr C. Sardet (14) (Institut de Génétique Moléculaire, Montpellier, France). The full length E2F4 cDNA was subcloned into a pLVX-Tight-Puro (Clontech, CA, USA) expression vector. PCR was performed to insert the HA-tag using oligonucleotides containing the HA-tag and a KOZAK sequence: 5′-A GAC TAG GAT CC C ACC ATG TAT GAT GTT CCT GAT TAT GCT AGC CTC CCG GCG GAG GCC GGG CCA CAG GCG CCG-3′ and 5′-TCT GTA CTC GAG TCA GAG GTT GAG AAC AGG CAC ATC AAA GAG GTC-3′. PCR products were next digested and ligated in BamHI/EcoRI-digested pLVX-Tight-Puro vector. E2F4 mutants (Ser)₁₂ and (Ser)₁₄ were obtained by site-directed mutagenesis using the following primers, respectively: 5′-CTG GAC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AAC AGT AAC-3′ and 5′-GTT ACT GTT GCT GCT GCT GCT GCT GCT GCT GCT GCT GCT GCT GCT GTC CAG-3′; 5′-CTG GAC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AAC AGT AAC-3′ and 5′-GTT ACT GTT GCT GCT GCT GCT GCT GCT GCT GCT GCT GCT GCT GCT GCT GCT GTC CAG-3′. pBABE-Puro HA-wtE2F4, HA-E2F4(Ser)₁₂ and HA-E2F4(Ser)₁₄ vectors were obtained by subcloning from pLVX-Tight-Puro vectors (BamHI/EcoRI). The pCMVHA-DP2 expression vector was obtained from Dr J.A. Lees (15) (Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA).

Human embryonic kidney 293 cells and all colorectal cancer cell lines were obtained from ATCC (Manassas, VA, USA). 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Burlington, ON, Canada) containing 10% FBS supplemented with 2 mM glutamine, 10 mM HEPES, 0.5 IU/ml penicillin and 50 μg/ ml streptomycin (all obtained from Wisent, St-Bruno, QC, Canada). The human colorectal cancer cells were cultured in the following media: HT-29 and HCT116 in McCoy’s 5A medium; DLD-1 and COLO205 in RPMI-1640 medium; LoVo in F-12K medium and Caco-2/15 in DMEM medium. All of the above media were supplemented with 10% FBS, 2 mM glutamine, 10 mM HEPES, 0.5 IU/ml penicillin and 50 μg/ml streptomycin. Non-immortalized human intestinal epithelial cells (HIEC) were isolated by Perreault and Beaulieu (16) and cultured in Opti-MEM (Invitrogen) medium supplemented with 2 mM glutamine (Invitrogen), 5% fetal bovine serum (FBS), 10 mM HEPES, 0.5 IU/ml penicillin, 50 μg/ml streptomycin and 0.2 IU/ml insulin (Connaught Novo Laboratories, Willowdale, Canada). HIEC, originally generated from normal human fetal small intestine at mid-gestation, express typical features of the lower adult crypt region and are unable to differentiate (16).

E2F4 retroviruses were produced by co-transfecting pBabe-puro HA-wtE2F4, (Ser)₁₂ or (Ser)₁₄ constructs with the pVPack vector system (Agilent Technologies, Mississauga, ON, Canada) in 293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s recommendations. Viral supernatants were harvested 48 h following transfection, filtered through a 0.45-μm filter and cells were infected following the instructions of Agilent Technologies.

Cells were washed twice with ice-cold PBS, then lysed in Triton lysis buffer [1% Triton X-100, 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM EDTA, 5% glycerol, 40 mM β-glycerophosphate, 50 mM NaF, 200 μM orthovanadate, 5% pepstatin, 5% aprotinin, 5% leupeptin and 5% phenylmethylsulfonyl fluoride (PMSF)] for 30 min under light agitation. Lysates were then cleared by centrifugation (15,000 g, 10 min) and 4X Laemmli buffer (2.3% SDS, 10% glycerol, 0.005% bromophenol blue and 5% β-mercaptoethanol) was added to supernatants for gel analysis. Whole cell extracts were separated on 10% SDS-PAGE gels and then electro-transferred onto polyvinylidene fluoride (PVDF) membranes (Perkin-Elmer, Montréal, Québec, Canada). Membranes were blocked for 1 h at 20°C using 0.05% Tween/PBS containing 5% non-fat dry milk then incubated overnight in primary antibodies diluted in blocking solution. Membranes were next incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (GE Healthcare, Baie d’Urfé, Canada) in blocking solution for 1 h. The blots were visualized using homemade ECL (Tris-HCl 100 mM pH 8.5, 1.25 mM luminol, 225 μM coumaric acid and 2.9 mM H₂O₂). Protein concentrations were measured using the BCA procedure (Thermo Scientific, Waltham, MA, USA) as described by the manufacturer, with bovine serum albumin (BSA) as standard.

293T cells were co-transfected with pLVX-Tight-Puro empty vector, HA-wtE2F4, HA-E2F4(Ser)₁₂ or HA-E2F4(Ser)₁₄ and pLVX Tet-Off Advanced using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Cells were cultured in absence of tetracycline to allow E2F4 expression. For stability assays, cells were transfected for 24 h then treated with cycloheximide for the indicated times. For luciferase assays, 293T cells were also transfected with pE2F-TA-Luc luciferase reporter vector (Clontech), pRL-SV40 Renilla luciferase reporter (Promega, Madison, WI, USA) and pCMV-HA-DP2 vector. Forty-eight hours following transfection, luciferase activity was measured using the Dual-Luciferase^® Assay Reporter System (Promega) according to the manufacturer’s protocol. Relative E2F4 luciferase activity was normalized using the Renilla reporter to account for transfection efficiency. For co-immunoprecipitation assays, cells were transfected for 48 h and lysed with Triton lysis buffer. Cleared lysates were incubated with anti-E2F4 antibody (3 h, 4°C), after which protein A Sepharose CL-4B beads (GE Healthcare) were added for an additional hour. Immunocomplexes were washed three times with Triton X-100 lysis buffer then eluted with Laemmli buffer and loaded on SDS-PAGE gels. Extensive protocols of 293T cells transfection and luciferase assays are available upon request.

Genomic DNAs (gDNAs) were isolated from the human colorectal cancer cells Caco-2/15, COLO205, HT-29, LoVo, DLD-1, HCT116 et SW48 as well as from normal HIEC using the Spin Doctor Solution Set (Gerard Biotech, Oxford, OH, USA) according to the manufacturer’s protocol. The E2F4 microsatellite was amplified by PCR from obtained gDNAs and PCR products were analyzed by capillary electrophoresis by the Laboratoire de Génomique Fonctionnelle de l’Université de Sherbrooke in order to determine the number of CAG repeats in the E2F4 microsatellite in each of the intestinal cell lines using control plasmid DNA with 12, 13 or 14 CAG repeats [pLVX-Tight-Puro E2F4 (Ser)₁₂, wtE2F4 and E2F4(Ser)₁₄, respectively]. The Agilent 2100 Bioanalyzer (Agilent Technologies) and the LabChip^® 90 (Caliper, Hopkinton, MA, USA) were used according to the manufacturer’s recommendations.

Total RNA was isolated with the RNeasy mini kit (Qiagen, Mississauga, ON, Canada). RT-PCR was performed using avian myeloblastosis virus reverse transcriptase (Roche Diagnostics) and conventional PCR analysis was conducted using Taq DNA polymerase according to the manufacturer’s instructions (Qiagen). Real-time PCR analyses were performed with a LightCycler apparatus (Roche Diagnostics). Experiments were executed and analyzed with the LightCycler software 4.0 according to the manufacturer’s recommendations. Synthesis of double-stranded DNA during PCR cycles was monitored with SYBR Green I (QuantiTect SYBR Green PCR kit; Qiagen). All samples were processed in triplicate. E2F4 expression was quantified relative to β2-microglobulin expression. A standard calibration curve was prepared for each gene by using serial dilutions of the calibrator sample, and crossing point values were plotted vs. the log of the relative concentration of each dilution. This standard curve was used to correct for differences in PCR efficiencies. Oligonucleotide primers used for DNA amplification were synthesized by Integrated DNA Technologies (San Diego, CA, USA). Primer sequences are available upon request.

RPMI medium (Wisent, QC, Canada) was complemented with 20% FBS, 4 mM glutamine, 20 mM HEPES, 1 IU/ml penicillin and 100 μg/ml streptomycin. This pre-warmed medium was mixed 1:1 (v/v) with autoclaved 1.4% agarose type VII maintained at ∼42°C and 6-well dishes were pre-coated with 1.5 ml/well. A total of 30,000 cells/well were added to the RPMI/agarose mixture. Plates were allowed to solidify then placed at 37°C under 5% CO₂. Fresh RPMI supplemented with 10% FBS was added on the surface of the agarose every 2–3 days. After 2–3 weeks, colonies were stained by adding 1 ml of PBS containing 0.5 mg/ml MTT to each well and incubated 2 h at 37°C and 5% CO₂. Images were acquired using an AlphaImager camera (Alpha Innotech Corp.). Colonies were counted and their size assessed using ImageJ software.

Assays were performed in triplicate. Typical western blots shown are representative of three independent experiments. Densitometric analyses were performed using ImageJ software.

To determine the impact of serine stretch-associated mutations on E2F4 function, both mutant forms of E2F4 reported to be frequently observed in CRC were generated, namely E2F4(Ser)₁₂ and E2F4(Ser)₁₄. These mutants as well as the wild-type form of E2F4 were then cotransfected along with thymidine kinase luciferase reporter gene. Previous studies have reported that the gene encoding for this protein contains E2F4-responsive elements in their promoters (17–19) and our previous studies have shown that E2F4 silencing in HIEC markedly reduced thymidine kinase mRNA levels (8). As shown in Fig. 1A, both E2F4(Ser)₁₂ and E2F4(Ser)₁₄ mutants exhibited significant enhanced transcriptional activities relative to thymidine kinase gene compared with wtE2F4. The increased transcriptional activity of mutants versus wild-type E2F4 prompted us to verify their mRNA and protein expression by quantitative PCR and western blot analyses respectively. As shown in Fig. 1B, protein levels of E2F4(Ser)₁₂ and E2F4(Ser)₁₄ mutants were consistently higher than levels of wild-type E2F4. Of note however, these increased protein expression levels of mutants could not be attributed to an increase in mRNA levels which were similar to those of wild-type E2F4 (Fig. 1C).

Since increased protein but not mRNA levels were observed for E2F4(Ser)₁₂ and E2F4(Ser)₁₄ mutants, this suggests that colorectal cancer-associated mutations may affect protein stability of E2F4. Therefore, 293T cells were transiently transfected with either wild-type E2F4, E2F4(Ser)₁₂ mutant or E2F4(Ser)₁₄ mutant and then treated with cycloheximide to inhibit protein synthesis. Thereafter, cells were lyzed at different time intervals in order to analyze protein expression levels of E2F4 forms. As shown in Fig. 2A (lanes 2 versus 7 and 12), expression of E2F4(Ser)₁₂ and E2F4(Ser)₁₄ mutants was higher than wild-type E2F4 expression in untreated cells (time 0). Following cycloheximide treatment however, E2F4(Ser)₁₂ and E2F4(Ser)₁₄ protein levels decreased much more slowly than that of wild-type E2F4 (Fig. 2A and B, for densitometric analysis). Specifically, 3 h after cycloheximide addition, expression of E2F4 protein was drastically decreased while expression of E2F4(Ser)₁₂ and E2F4(Ser)₁₄ mutants remained at control (time 0) levels. Accordingly, the half-life of wild-type E2F4 protein was ∼5 h whereas E2F4(Ser)₁₂ and E2F4(Ser)₁₄ half-lives were >12 h.

Furthermore, association of E2F transcription factors with a pocket protein has been reported to protect E2F factors against degradation (20–22). We therefore analyzed whether colorectal cancer-associated mutations modulate E2F4 interaction with p130/RBL2, the main pocket protein partner for E2F4 in various cell types including intestinal epithelial cells (9). Indeed, association of E2F4 with p130 was found to promote E2F4 stability (20). Thus, cells were co-transfected with p130 along with wild-type E2F4, E2F4(Ser)₁₂ or E2F4(Ser)₁₄ after which E2F4 was immunoprecipitated and the amounts of associated p130 analyzed by western blotting. As shown in Fig. 2C, neither E2F4(Ser)₁₂ nor E2F4(Ser)₁₄ were found to have an altered capacity to associate with p130 in comparison to wild-type E2F4 (when normalized to immunoprecipitated E2F4 levels). These results suggest that the colorectal cancer-associated E2F4 mutants are more stable than wild-type E2F4, independently of their capacity to interact with p130.

The CAG triplet repeat in the coding region of the E2F-4 gene has been reported to be mutated in colorectal cancers exhibiting a microsatellite instability (MSI) phenotype. We therefore analyzed E2F4 gene and protein expression patterns in a panel of colorectal cancer cell lines which are microsatellite unstable (MSI) (HCT116, SW48, DLD-1, LoVo) and microsatellite stable (MSS) (COLO205, Caco-2/15 and HT-29) (23,24). As shown in Fig. 3A and B, E2F4 mRNA and protein levels were observed to be globally enhanced in colorectal cancer cell lines exhibiting microsatellite instability.

Given this increased E2F4 expression in MSI colorectal cancer cells, the number of CAG repeats in the E2F4 microsatellite was determined in each cell line by capillary electrophoresis as described in Materials and methods. Analysis of the 13-serine residue microsatellite of E2F4 revealed that only the SW48 cell line expressed mutated E2F4 with a deletion of one CAG repeat causing a deletion of one serine residue. Interestingly, amongst all colorectal cancer cell lines analyzed, SW48 exhibited the highest expression of E2F4 protein (Fig. 3A). In order to associate this increased protein expression with increased E2F4 stability, endogenous E2F4 half-life was analyzed in SW48 cells comparatively to Caco-2/15, a MSS cell line in which E2F4 is wild-type. SW48 and Caco-2/15 cells were treated with cycloheximide to inhibit protein synthesis for various time intervals. As shown in Fig. 3C and D, following cycloheximide treatment, E2F4 protein levels decreased more rapidly in Caco-2/15 compared to SW48 cells. Specifically, E2F4 half-life in Caco-2/15 cells was established at 19 h whereas E2F4 half-life in SW48 cells was ∼48 h. These results confirm that the deletion of one serine residue in the serine stretch increases E2F4 stability resulting in increased protein levels.

Our previous report indicated that E2F4 transcription factor expression is required for anchorage-dependent and anchorage-independent growth of colorectal cancer cells (8). Because anchorage-independent growth potential may better correlate with tumorigenic growth in vivo, we determined whether E2F4 mutations in the serine stretch was correlated with stimulation of tumor cell growth in soft agar. The DLD-1 cell line was chosen because this line exhibits microsatellite instability and expresses moderate levels of endogenous wild-type E2F4. As shown in Fig. 4, expression of E2F4(Ser)₁₂ or E2F4(Ser)₁₄ mutants (Fig. 4A) increased the capacity of DLD-1 cells to form colonies in soft agar (Fig. 4B). Moreover, colonies formed following the expression of E2F4 mutants were significantly larger than colonies formed following the expression of wild-type E2F4 (Fig. 4C). Of note, similar results were obtained with the MSS cell line Caco-2/15 (data not shown).