Supplementary materials and methods

Immunohistochemistry

Human samples and mice xenograft tissue samples were fixed in 10% neutral buffered formalin and paraffin-embedded. Four µm sections were cut and deparaffinized in Histoclear. Heat Induced Epitope Retrieval (HIER) was performed on sections utilizing a pressurized de-cloaking chamber (Biocare Medical, LLC, Concord, CA, USA) and incubated in citrate buffer (pH 6.0) at 990C for 18 min. The sections were then washed thrice with PBS and endogenous biotin activity was blocked using Avidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA, USA) according to manufacturer’s instructions. Further, endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in methanol. The sections were blocked with 10% normal serum for 30 min to prevent non-specific binding and then incubated overnight with the primary antibodies (1:100) for WIF1, β-catenin, TCF-4, E-cadherin, cyclin D1, WNT1, p53, p21, VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase-3 (Cell Signaling Technology, Danvers, MA, USA), c-myc and CD31 (BD Biosciences, NJ, USA), and CD44 (Calbiochem). The biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) were used at a dilution of 1:400. The slides were further processed by using a Vectastatin ABC kit (Vector Laboratories, Burlingame, CA, USA). Antibody binding was visualized using 3,3'-diaminobenzidine (Sigma-Aldrich) as chromogen. Nuclei were counterstained with Mayer’s haematoxylin. A negative control sample was included in each run by omitting the primary antibody. For statistical analysis, the intensity of WIF1 immunostaining was scored as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong) in human tissues.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed in tissue sections using ApopTag peroxidase in situ apoptosis detection kit (Chemicon Int., Temecula, CA, USA) according to the manufacturer’s instructions. Briefly, sections were deparaffinized, rehydrated and incubated with Proteinase K for 20 min at 37° C and then washed with PBS. The endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min at room temperature. After washing with PBS, the sections were incubated with working concentration of terminal deoxynucleotidyl transferase (TdT) at 37° C for 1 h. The sections were then washed with PBS and finally incubated with anti-digoxigenin conjugate (peroxidase) at room temperature, and the signal was visualized with diaminobenzidine as chromogen.

Mitotic activity was evaluated directly by counting mitotic figures on 10 randomly selected fields of H&E stained tumor sections. Mitotic activity is expressed as a ratio of number of mitoses per high power field (200X). Microvessel density was measured based on the number of blood vessels stained for CD31 by analyzing 10 randomly selected fields (400X).

Immunofluorescence

The tissue sections were processed as explained under immunohistochemistry and the sections were then sequentially exposed to Phospho-Histone H3 antibody (Upstate) for 1 h at 30◦C and its appropriate secondary fluorescent conjugate alexa fluor 568 (red) (Invitrogen) for 30 min at room temperature. Subsequently, fluorescein conjugated TUNEL staining was performed using ApopTag in situ apoptosis detection kit (Chemicon Int) according to the manufacturer’s instructions. The slides were then wet-mounted and counterstained with Hoechst 33342 (Invitrogen).

Microscopic examination

Tissue sections were examined using Nikon 80i microscope base. For bright field, 10X or 60X digital images were taken with PlanAPO objectives and DXM1200C camera (Nikon, Melville, NY, USA). Fluorescent images were taken with 20X PlanFluoro objective utilizing CoolSnap ES2 camera (Photometrics, Tucson, AZ, USA). All images were captured utilizing NIS-Elements software (Nikon, Melville, NY, USA).

Cell lines

HeLa and C33A human cervical cancer cell lines were obtained from the American Tissue Culture Collection (ATCC) and maintained as recommended. The CC1 cell line was obtained from Dr. Lamonis A. Laimins and maintained as reported (Rader et al., 1990).

Treatment with 5-aza-2'-deoxycytidine

Cervical cancer cell lines were treated with the demethylating agent 5-aza-2'-deoxycytidine (50 µM) for 4 days. Total RNA, genomic DNA and protein were isolated and subjected to real-time reverse transcriptase-polymerase chain reaction (RT-PCR), methylation studies (methylation-specific PCR and bisulfite sequence analysis) and western blot analysis, respectively.

Real-time RT-PCR analysis

Total RNA was isolated from cells, microdissected human cervical normal and cancer tissues, and tumor xenograft tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and subjected to reverse transcription with SuperscriptTM II RNase H - Reverse Transcriptase and random hexanucleotide primers (Invitrogen, Carlsbad, CA, USA). The cDNA was subsequently used for real-time RT-PCR by SYBR chemistry (SYBR® Green I; Molecular Probes, Eugene, OR, USA) using gene specific primers (Supplementary Table 1) and Jumpstart Taq DNA polymerase (Sigma-Aldrich, St. Louis, MO, USA). The crossing threshold value determined by real-time RT-PCR was noted for the transcripts and normalized with b-actin. The changes in mRNA were expressed as fold change relative to control with ± SD value.

Methylation-specific PCR (MSP)

Genomic DNA was extracted from normal and cervical cancer samples, and cervical cancer cell lines as we previously described (Queimado et al., 1998). Bisulfite modification of genomic DNA was carried out using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA). Bisulfite-modified genomic DNA was amplified using primers that were specific for methylated (M) and unmethylated (U) DNA. The sequences of the M-specific primers were 5’-GGGCGTTTTATTGGGCGTAT-3’ (forward) and 5’-AAACCAACAATCAACGAAAC-3’ (reverse). The sequences of the U-specific primers were 5’-GGGTGTTTTATTGGGTGTAT-3’ (forward) and 5’-AAACCAACAATCAACAAAAC-3’. For sequencing analysis, bisulfite-modified genomic DNA was amplified using primers 5’-GAGTGATGTTTTAGGGGTTT-3’ (forward) and 5’-CCTAAATACCAAAAAACCTAC-3’ (reverse), designed to amplify nucleotides -555 to -140 of the WIF1 promoter region as previously described (Mazieres et al., 2004). Following PCR, the products were run in 1% agarose, then cut, blunted by standard protocol and cloned into pJET 1.2 vector. At least 5 random clones were selected for colony PCR and sequenced.

WIF1 construct

WIF1 was amplified from testis cDNA with the primers 5’-GAGATCTCTCGAGAGGAGGTCCTGAGCAGCATG-3’ (BglII-XhoI-2-WIF1-F) and 5’-TACCGCGGCCGCTAATGGTGATGGTGATGGTGCCAGATGTAATTGGATTCAGGTG-3’ (WIF1-R2-H6.NotI-KnpI), cloned in pGEM-T-EASY vector and sequenced. The pGEM-T WIF1 insert was then digested with XhoI and NotI, cloned into pCI-blast XhoI and NotI sites. Insertion sites were confirmed by sequencing.

Transfection and proliferation assay

Exponentially growing HeLa cells were plated onto 96-well plates at the density of 1×104 cells/well and transfected simultaneously with either pCI blast (empty vector) or pCI blast-WIF1 using LipoD293 transfection reagent (SignaGen Laboratories, Ijamsville, MD, USA). Cell proliferation was assessed at different time intervals (24, 48 and 72 h) by hexosaminidase assay (Landegren, 1984).

Cell cycle analysis

HeLa cells (3 x 105 cells/well) were plated onto six-well plates and simultaneously transfected with either empty vector or pCI blast-WIF1 using LipoD293, and cell cycle analysis was performed as we described previously (Natarajan et al., 2008). Briefly, 72 h post-transfection, the floating and attached cells were collected and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were washed and suspended in PBS. Single-cell suspension was fixed in ice-cold 70% ethanol for 2 h at 4°C. The cells were centrifuged to remove the ethanol and washed with PBS. The fixed cells were then incubated with 1 mg/ml propidium iodide (Sigma-Aldrich St. Louis, MO, USA), 0.1% Triton X-100 (Sigma-Aldrich) and 2 µg DNase-free RNase (Sigma-Aldrich) in PBS for 30 min at room temperature in dark. Flow cytometry was done with a FACSCalibur analyzer (Becton Dickinson, Mountain View, CA, USA), capturing 50,000 events for each sample. Results were analyzed with ModFit LT software (Verity Software House, Topsham, ME, USA).

Caspase-3/7 activity

HeLa cells (1 x 104 cells/well) were plated onto 96-well black plates and transfected simultaneously with either empty vector or pCI-blast-WIF1. Caspase-3/7 activity was measured 72 h post-transfection using the Apo-one Homogeneous Caspase-3/7 Assay kit as per the manufacturer’s instructions (Promega, Madison, WI, USA).

Western blot analysis

Cells and xenograft tumor samples were homogenized in cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA) and further sonicated and centrifuged at 12,000 rpm for 10 min at 4°C. Fifty µg proteins per lane were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). The membranes were blocked for 1 h with 5% non-fat milk and incubated overnight with primary antibodies [active β-catenin (Upstate, Millipore, Temecula, CA, USA), TCF-4 (Abcam, Cambridge, MA, USA), WIF1, Bax, Bcl-2 and Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA)]. This was followed by incubation with secondary antibodies coupled with HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. The membrane was then washed thrice in TBS-T. Immunoreactive antibody–antigen complexes were visualized with the enhanced chemiluminescence reagents (Thermo Scientific, Rockford, IL, USA).

Statistical analysis

Statistical analyses were performed using SAS® STAT Version 9.1 (SAS Institute Inc.). Independent means were compared using unpaired Student's t tests whose degrees of freedom were corrected, when appropriate, for inequality of variance. Data are expressed as mean ± s.d. for at least three independent experiments. We considered a P 0.05 to be statistically significant.

References

Landegren U. (1984). Measurement of cell numbers by means of the endogenous enzyme hexosaminidase. Applications to detection of lymphokines and cell surface antigens. J Immunol Methods 67: 379-388.

Mazieres J, He B, You L, Xu Z, Lee AY, Mikami I et al. (2004). Wnt inhibitory factor-1 is silenced by promoter hypermethylation in human lung cancer. Cancer Research 64: 4717-4720.

Natarajan G, Ramalingam S, Ramachandran I, May R, Queimado L, Houchen CW et al. (2008). CUGBP2 downregulation by prostaglandin E2 protects colon cancer cells from radiation-induced mitotic catastrophe. Am J Physiol Gastrointest Liver Physiol 294: G1235-1244.

Queimado L, Reis A, Fonseca I, Martins C, Lovett M, Soares J et al. (1998). A refined localization of two deleted regions in chromosome 6q associated with salivary gland carcinomas. Oncogene 16: 83-88.

Rader JS, Golub TR, Hudson JB, Patel D, Bedell MA, Laimins LA. (1990). In vitro differentiation of epithelial cells from cervical neoplasias resembles in vivo lesions. Oncogene 5: 571-576.

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