Thirty-five human cell lines were used, derived from the following tissue or tumor sources: 293FT, embryonal kidney; K562, chronic myelogenous leukemia; HMMME, ACC-MESO-1 and ACC-MESO-4, mesothelioma; JHOS-2 and JHOS-3, ovarian serous adenocarcinoma; HeLa, uterine cervical cancer; TE-2, TE-4, TE-5, TE-8, TE-9, TE-14, SGF7 and HEC46, esophageal cancer; TFK-1, HuCCT1, TKKK, RBE and HuH-28, bile duct cancer; PANC-1, PCI-6, KP-1N, SUIT-2, AsPC-1 and BxPC-3, pancreatic cancer; A549, RERF-LC-MS, RERF-LC-OK, ABC-1 and VMRC-LCD, lung adenocarcinoma; H226, LK-2 and PC10, lung squamous cell carcinoma.

293FT was purchased from Invitrogen (Carlsbad, CA, USA). K562, HMMME, ACC-MESO-1 (37), ACC-MESO-4 (37), JHOS-2, JHOS-3, HeLa, TFK-1, HcCCT1, TKKK, RBE, HuH-28 and PANC-1 were purchased from the RIKEN BioResource Center Cell Bank (Tsukuba, Japan). The TE series was provided by Dr T. Nishihira (Tohoku University, Sendai, Japan) (38). SGF7 was provided by Dr T. Saito (Toyama Medical and Pharmaceutical University, Toyama, Japan) (39). HEC46 was provided by Dr T. Toge (Hiroshima University, Hiroshima, Japan) (40). PCI-6 was provided by the First Department of Pathology, Hokkaido University (Sapporo, Japan) (41). KP-1N, SUIT-2, A549, RERF-LC-MS, RERF-LC-OK, ABC-1, VMRC-LCD, H226, LK-2 and PC10 were purchased from the Japanese Cancer Research Resource Bank (Tokyo, Japan). AsPC-1 and BxPC-3 were provided by the American Type Culture Collection (ATCC; Manassas, VA, USA).

293FT was cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO, USA). The other cell lines were cultured in RPMI-1640 (Sigma-Aldrich). Each medium was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO₂. Trypsin (0.25%) was used for subculture.

Cancer tissues and corresponding normal epithelial tissues were obtained from 552 patients with esophageal (101 patients), bile duct (96 patients), pancreatic (99 patients), and lung cancer (256 patients) who underwent resection in the Department of Gastroenterological Surgery II, Hokkaido University, Japan between 1994 and 2005. These tissue samples were fixed in 10% formalin and embedded in paraffin blocks. Three or four spots of each tissue, of uniform 0.6-mm diameter, were punched out and consolidated into 25 paraffin blocks using a Manual Tissue Arrayer (Beecher Instruments, Inc., Sun Prairie, WI, USA); the resultant 25 blocks were used for immunohistochemistry. As possible positive controls for WT1 expression in tissue samples, kidney, pleura, testis, pleural mesothelioma and ovarian serous adenocarcinoma were also examined under the same conditions.

Negative and positive controls for WT1 protein expression were generated as follows. First, internal ribosome entry site and complementary DNA (cDNA) encoding green fluorescent protein (GFP) were cloned into the multiple cloning site of plasmid vector pcDNA3.1(+) (Invitrogen); the resultant plasmid is termed empty vector. WT1 cDNA was amplified by polymerase chain reaction (PCR), using human kidney QUICK-Clone cDNA (Takara, Otsu, Japan) as a template and cloned into empty vector. The resultant plasmid vector is termed vector WT1. The base sequence of vector WT1 was confirmed by the single-nucleotide primer extension method. Empty vector and vector WT1 were transfected into 293FT and HeLa cells using Lipofectamine 2000 (Invitrogen). Successful transfection was verified by observing GFP expression. Cells transfected with empty vector and vector WT1 were, respectively, used as negative and positive controls for WT1 protein expression. The WT1 mRNA contains two splice sites that can be skipped during RNA splicing; consequently, the WT1 gene encodes four variant isoforms of WT1 protein (Fig. 1). We constructed the corresponding four types of positive controls for WT1 protein expression.

GFP cDNA was cloned into the multiple cloning site of pcDNA3.1(+); the resultant plasmid is termed vector GFP. WT1 cDNA was cloned into vector GFP such that the WT1 cDNA sequence was fused to the C-terminus of the GFP cDNA, which lacked a stop codon; the resultant plasmid is termed vector GFP-WT1. The base sequence of vector GFP-WT1 was confirmed by the single-nucleotide primer extension method. Vector GFP and vector GFP-WT1 were transfected into 293FT and HeLa cells using Lipofectamine LTX (Invitrogen). Cells transfected with vector GFP and vector GFP-WT1 were predicted to produce GFP and fusion protein GFP-WT1, respectively. As noted above, the WT1 mRNA has four splicing variants (Fig. 1); therefore, we constructed the corresponding four types of vector GFP-WT1 plasmids.

Cultured cells which were detached using 0.25% trypsin and sedimented by centrifugation or excised tissue samples were fixed in 10% formalin and embedded in paraffin blocks. Thin sections (2 μm thick) were de-waxed and rehydrated, and antigens were retrieved in pressure vessels under the following conditions: citrate buffer (pH 7.0), 2 atm, 100°C, 2 min. For additional antigen retrieval, sections for WT1 antibody 6F-H2 were incubated for 5 min at room temperature with 10% proteinase K ready-to-use enzyme (Dako, Kyoto, Japan) before treatment in the pressure vessels. The sections were immersed for 15 min at room temperature in 0.3% H₂O₂ diluted with methanol, in order to block endogenous peroxidase activity, and then incubated in 10% normal goat serum (Nichirei Corp., Tokyo, Japan) at room temperature for 30 min to reduce non-specific binding. WT1 antibodies 6F-H2 (mouse monoclonal; Dako), ab89901 (rabbit monoclonal; Abcam, Cambridge, UK) and C-19 (rabbit polyclonal; Santa Cruz Biotechnology, Dallas, TX, USA), used as primary antibodies (Fig. 1), were diluted 1:100, 1:200 and 1:100, respectively, with antibody diluent (Dako). The sections were incubated with the diluted primary antibodies at 4°C overnight. Mouse IgG1 (Dako) and rabbit polyclonal IgG (Abcam) were used for negative controls against samples with primary antibodies. Next, sections were incubated for 30 min at room temperature with biotinylated goat antibody to mouse and rabbit immunoglobulin (Histofine Simple Stain MAX PO MULTI; Nichirei Corp.). Finally, sections were stained with 3-3′-diaminobenzidine tetrahydrochloride (Histofine Simple Stain DAB Solution; Nichirei Corp.) and then lightly counter-stained by hematoxylin.

In cell lines, immunostaining scores for the entire nucleus, the nuclear bodies, and the cytoplasm were calculated independently. Immunostaining intensities of entire nucleus and cytoplasm in individual cells were classified into four grades and intensity of nuclear bodies was classified into two grades as shown in Fig. 2. Immunostaining scores for samples were calculated as the sum of the immunostaining intensities of individual cells, divided by the total number of the cells; thus, the score represents the average immunostaining intensity of all cells in the sample. Immunostaining scores were independently calculated by two surgeons under the guidance of a pathologist and the average value was regarded as the final immunostaining score.

Total protein was extracted from cultured cells using triple-detergent lysis buffer. Protein concentration was measured by the Bradford method using a commercial protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Protein was boiled for 2 min for antigen retrieval, subjected to SDS-PAGE, and blotted onto a Hybond-ECL nitrocellulose membrane (GE Healthcare Life Sciences, Piscataway, NJ, USA). The membrane was blocked at 4°C overnight with 5% skim milk, incubated with diluted primary antibodies at room temperature for 1 h, and then incubated with diluted secondary antibodies at room temperature for 1 h. WT1 antibodies 6F-H2, ab89901 and C-19 (Fig. 1), mouse monoclonal GFP antibody (Clontech Laboratories, Inc., Otsu, Japan) and actin C4 antibody (Millipore, Billerica, MA, USA) were used as primary antibodies. Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG H+L antibodies (Jackson ImmunoResearch, West Grove, PA, USA) or peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG H+L antibodies (Jackson ImmunoResearch) were used as secondary antibodies. Protein bands were visualized using the ECL-Plus Western blotting detection system (GE Healthcare Life Sciences) and Detector Lumino Imaging Analyzer model FAS-1000 (Toyobo, Co., Ltd., Osaka, Japan). Intensities of protein bands were quantitated using ImageJ (http://rsbweb.nih.gov/ij/), corrected for the intensities of the corresponding actin bands, and normalized to the intensity of the protein band obtained from vector WT1[−−] transfectants (defined as 1). Two patterns of experimental conditions, which differed in the amount of protein applied to each gel lane and the dilution factors of the antibodies (Table I), were used. However, the intensities of protein bands were compared under the same conditions.

Total RNA was extracted from cultured cells using the TRI reagent (Sigma-Aldrich). Possible DNA contamination was eliminated using RQ1 RNase-Free DNase (Promega, Tokyo, Japan). After the DNase treatment, the absence of DNA contamination was confirmed by PCR using the primers for β-actin (described below). cDNA was synthesized from mRNA by reverse transcription reaction using the SuperScript VILO cDNA Synthesis kit (Invitrogen). cDNA diluted 20-fold with double-distilled water was used as a template for quantitative PCR. Quantitative PCR was performed by two methods: the intercalator-based method using Power SYBR-Green PCR Master Mix (Applied Biosystems, Tokyo, Japan) and the fluorescent probe-based method using TaqMan Universal Master Mix II (Applied Biosystems). Reaction conditions consisted of 40 cycles of 94°C for 30 sec, 60.4°C for 30 sec and 72°C for 30 sec. All reactions were performed in triplicate. The results of quantitative PCR were analyzed on an ABI PRISM 7000 Sequence detection system (Applied Biosystems). Expression levels of WT1 mRNA were normalized to the corresponding β-actin mRNA and relativized considering those of WT1 mRNA of K562 as 1.

The base sequences of primers and internal probes were as follows: WT1 sense primer, 5′-TGCGGAGCCCAATACAGAATACAC-3′ and WT1 reverse primer, 5′-TCAGATGCCGACCGTACAAGAG-3′; WT1 internal probe, 5′-FAM-AGAGGCATTCAGGATGTGCGACG-TAMRA-3′; β-actin sense primer, 5′-CAACCGCGAGAAGATGACCC-3′ and β-actin reverse primer, 5′-ACCGGAGTCCATCACGATGC-3′; β-actin internal probe, 5′-FAM-CCAGGCTGTGCTATCCCTGTACGC-TAMRA-3′. The primers for WT1 were designed to bind to WT1 cDNA between exons 6 and 7, so that the four spliced variants of WT1 cDNA were all recognized (Fig. 1). The primers for β-actin were designed to bind to β-actin cDNA between exons 3 and 4.

Correlation of paired quantitative variables was evaluated by the Spearman rank method using StatView version 5.0 software (SAS Institute, Inc., Cary, NC, USA). Differences were considered significant when r >0.4 and P<0.05.

We transfected 293FT and HeLa with vectors encoding the four splice variants of WT1 (Fig. 1). WT1-transfected cells were expected to produce excessive exogenous WT1 protein. As shown in Fig. 3, western blotting with two WT1-specific monoclonal antibodies (6F-H2 and ab89901) and one WT1-reactive polyclonal antibody (C-19) yielded clear 50–55-kDa bands in cells expressing any of the four WT1 variants, even though the antibodies recognize different sites (Fig. 1). Immunohistochemistry with these three antibodies resulted in strong staining in the nucleus of all WT1 transfectants, corresponding to the appearance of the 50–55-kDa bands in western blotting. In addition to the nuclear staining, these antibodies yielded modest cytoplasmic staining in various cells including empty-vector transfectants. Especially, C-19 yielded significant cytoplasmic immunostaining in all tested cells, regardless of their transfection status. In conclusion, enforced WT1 expression was detected in the nucleus of the two different cell lines by the three different antibodies. Polyclonal C-19 antibody may exhibit non-specific as well as specific reactivity. We used these transfectants as positive and negative controls for further analyses.

By itself, immunohistochemistry does not distinguish specific from non-specific staining, because it does not provide the information such as the molecular weights or sequences of the detected proteins. To verify the specificity for endogenous WT1 in immunohistochemistry, we compared results of WT1-immunohistochemistry with those of western blotting and qRT-PCR in 35 human cell lines. Fig. 4 shows the results of immunohistochemistry, western blotting and qRT-PCR. For immunohistochemistry, immunostaining scores were calculated independently in the entire nucleus, the nuclear bodies and the cytoplasm (Fig. 2). In western blotting, multiple protein bands with different molecular weights were observed; these bands were classified into A-G (Fig. 4B) and their intensities were, respectively, quantified. In qRT-PCR, we used both intercalator-based and fluorescent probe-based methods. Table II shows all quantified results: the immunostaining scores, the intensities of western blotting bands and WT1 mRNA levels.

Table III shows the relationships among quantified results from immunohistochemistry, western blotting and qRT-PCR of the 35 cell lines; positive correlations in significance (both r>0.4 and P<0.05) are indicated in bold. Any relationship among the intensities of western blot bands and WT1 mRNA expression levels yielded strongly positive correlations with statistical significance, with the exception of relationships including western blot band B. The immunostaining scores of the entire nucleus generated by monoclonal 6F-H2 positively correlated with any intensity of western blotting bands other than band B, as well as any WT1 mRNA expression levels. The immunostaining scores of the entire nucleus by monoclonal ab89901 positively correlated with the nuclear immunostaining scores by 6F-H2, intensities of multiple western blot bands, and WT1 mRNA expression levels. By contrast, the immunostaining scores of the entire nucleus by polyclonal C-19 did not correlate with intensities of western blot bands or WT1 mRNA expression levels. No significant correlations were observed between any immunostaining score of the nuclear bodies or cytoplasm generated by three WT1 antibodies and any result of western blotting or qRT-PCR.

These results suggest that western blot bands except for band B and WT1 mRNA expression levels are credible indicators of endogenous WT1 expression. In attunement with them, nuclear immunostaining using 6F-H2 and ab89901 seems to quantitatively reflect endogenous WT1 expression. By contrast, cytoplasmic immunostaining using WT1 antibodies does not specifically reflect WT1 expression because it did not have any significant association with results from other detection methods.

To verify the intracellular localization of WT1 protein by another method, we examined the localization of GFP-WT1 fusion protein. Fig. 5 shows microscopic images of cells transfected with vector GFP-WT1. While green fluorescence was observed in both the nucleus and the cytoplasm of cells transduced with GFP alone, it was observed only in the nucleus of cells transfected with vector GFP-WT1. Expression of GFP-WT1 fusion protein in the transfectants was confirmed by western blotting (Fig. 6). These results indicate that WT1 protein strongly prefers to be concentrated in the nucleus supporting the idea that nuclear immunostaining quantitatively reflects WT1 expression.

We examined WT1 expression in primary cancer tissue samples of esophagus, bile duct, pancreas and lung with the corresponding normal epithelial tissues by immunohistochemistry using 6F-H2, an antibody shown above to be appropriate for WT1-immunohistochemistry. As shown in Fig. 7 and Table IV, nuclear immunostaining was almost never detected in cancer cells or normal epithelium of esophagus, bile duct, pancreas, and lung, whereas it was firmly observed in mesothelioma, ovarian cancer and healthy tissues such as kidney and pleura.