Differential detection of cytoplasmic Wilms tumor 1 expression by immunohistochemistry, western blotting and mRNA quantification.

Wilms tumor 1 (WT1) is considered to be a promising target of cancer treatment because it has been reported to be frequently expressed at high levels in various malignancies. Although WT1-targeted cancer treatment has been initiated, conclusive detection methods for WT1 are not established. The present study aimed to consolidate immunohistochemistry for WT1 with statistical basis. Transfected cells with forced WT1 expression yielded specific western blot bands and nuclear immunostaining; cytoplasmic immunostaining was not specifically recognized. Immunohistochemistry, western blotting, and quantitative reverse transcriptase-polymerase chain reaction were performed in 35 human cell lines using multiple WT1 antibodies and their results were quantified. Relationships among the quantified results were statistically analyzed; the nuclear immunostaining positively correlated with western blot bands and mRNA expression levels, whereas cytoplasmic immunostaining did not. These results indicate that nuclear immunostaining reflects WT1 expression but cytoplasmic immunostaining does not. The nuclear immunostaining was barely (3/541) observed in primary cancer of esophagus, bile duct, pancreas and lung. Although the present study has some limitations, the results indicate that the cytoplasmic immunostaining does not correlate with actual WT1 expression and prompts researchers to carefully evaluate target molecule expression in treatment of cancer.

Soluble expression and purification of tumor suppressor WT1 and its zinc finger domain.

Full length murine WT1 and its zinc finger domain were separately inserted into Escherichia coli expression vectors with various fusion tags on either terminus by Gateway technology (Invitrogen) and expression of soluble protein was assessed. Fusion proteins including the four zinc finger domains of WT1 were used to optimize expression and purification conditions and to characterize WT1:DNA interactions in the absence of WT1:WT1 interactions. Zinc finger protein for in vitro characterization was prepared by IMAC purification of WT1 residues 321-443 with a thioredoxin-hexahistidine N-terminal fusion, followed by 3C protease cleavage to liberate the zinc fingers and cation exchange chromatography to isolate the zinc fingers and reduce the level of the truncated forms. Titration of zinc finger domain with a binding site from the PDGFA promoter gave a K(d) of 100±30nM for the -KTS isoform and 130±40nM for the +KTS isoform. The zinc finger domain was also co-crystallized with a double-stranded DNA oligonucleotide, yielding crystals that diffract to 5.5Å. Using protocols established for the zinc finger domain, we expressed soluble full-length WT1 with an N-terminal thioredoxin domain and purified the fusion protein by IMAC. In electro-mobility shift assays, purified full-length WT1 bound double-stranded oligonucleotides containing known WT1 binding sites, but not control oligonucleotides. Two molecules of WT1 bind an oligonucleotide presenting the full PDGFA promoter, demonstrating that active full-length WT1 can be produced in E. coli and used to investigate WT1 dimerization in complex with DNA in vitro.

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Marsupial WT1 has a novel isoform and is expressed in both somatic and germ cells in the developing ovary and testis.

The Wilms' tumour 1 gene is essential for the formation of the mouse and human urogenital systems. We characterised this gene and examined its expression throughout gonadal development in a marsupial, the tammar wallaby. WT1 protein was detected in the Sertoli and granulosa cells of the developing testis and ovary, respectively. There was also strong immunostaining in the germ cells of both males and females at all stages of gonadal development. In the adult gonads WT1 appears to be dynamically regulated during spermatogenesis and oogenesis. Tammar WT1 has a novel isoform in which a portion of exon 1 is removed, partially deleting the RNA recognition motif (RRM). Despite its removal, WT1 still localised to RNA rich regions of the oocyte including speckled bodies within the nucleus, in the nucleolus and the perinucleolar compartment. This suggests that the RRM is not required for WT1 co-localisation with RNA. This is also the first report of WT1 in association with the perinucleolar compartment, important for RNA metabolism. Our data suggest that WT1 has a conserved function in both the somatic and germ cell lineages of the gonads of marsupials.

Two methods for improved purification of full-length mammalian proteins that have poor expression and/or solubility using standard Escherichia coli procedures.

Many mammalian proteins are multifunctional proteins with biological activities whose characterization often requires in vitro studies. However, these studies depend on generation of sufficient quantities of recombinant protein and many mammalian proteins cannot be easily expressed and purified as full-length products. One example is the Wilm's tumor gene product, WT1, which has proven difficult to express as a full-length purified recombinant protein using standard approaches. To facilitate expression of full-length WT1 we have developed approaches that optimized its expression and purification in Escherichia coli and mammalian cells. First, using a bicistronic vector system, we successfully expressed and purified WT1 containing a C-terminal tandem affinity tag in 293T cells. Second, using a specific strain of E. coli transformed with a modified GST vector, we successfully expressed and purified N-terminal GST tagged and C-terminal 2x FLAG tagged full-length human WT1. The benefits of these approaches include: (1) two-step affinity purification to allow high quality of protein purification, (2) large soluble tags that can be used for a first affinity purification step, but then conveniently removed with the highly site-specific TEV protease, and (3) the use of non-denaturing purification and elution conditions that are predicted to preserve native protein conformation and function.

Expression, purification, and characterization of the 4 zinc finger region of human tumor suppressor WT1.

Wilm's Tumor gene 1 (WT1) encodes a zinc finger protein with four distinct splice isoforms. WT1 has a critical role in genesis of various cancer types both at the DNA/RNA and the protein level. The zinc-finger DNA-binding capacity of the protein is located in the C-terminal domain. Two recombinant proteins, 6HIS-ZN-wt1 and 6HIS-ZN+wt1, corresponding to two alternative splice variants of the C-terminal regions of human WT1 (-KTS) and WT1 (+KTS), respectively, were over-expressed with hexa-histidine fusion tags in inclusion bodies in Escherichia coli for crystallization studies. A combination of Ni2+-NTA affinity and size-exclusion chromatography was applied for purification of the proteins in denaturing conditions. The effects of various buffers, salts and other additives were scrutinized in a systematic screening to establish the optimal conditions for solubility and refolding of the recombinant WT1 proteins. Circular dichroism analysis revealed the expected betabetaalpha content for the refolded proteins, with a notable degradation of the alpha-helical segment in the DNA-free state. Electrophoretic mobility shift assay with double-stranded DNA containing the double Egr1 consensus site 5'-GCG-TGG-GCG-3' confirmed that 6HIS-ZN-wt1 has higher DNA binding affinity than 6HIS-ZN+wt1.

[Real-time PCR quantification of bcr/abl chimera and WT1 genes in chronic myeloid leukemia].

Quantification of mRNAs deriving from malignant cells is useful for estimating leukemic states. In this study, we have developed RT-PCR methods using real-time PCR detection system, a LightCycler, for quantification of bcr/abl chimerical genes in peripheral blood and bone marrow of chronic myeloid leukemia patients. Total amounts of RNA extracted were corrected using beta-actin gene as an internal standard. The coefficients of variation of intra-assay variation and inter-assay variation for each gene were within a range of 1.7-26.0% which showed more precise quantification than the competitive PCR method. The coefficients of variation of assay are within a range of 7.7-27.6% in the case of using three samples of normal subjects from blood collecting to quantification of bcr gene. Bcr/abl and WT1 genes could be measured from 10(2) to 10(8) copies and 10 to 10(5) copies with linearity, respectively. Using real-time PCR detection with LightCycler system, 2 x 10(3) K562 cells among 2 x 10(6) total cells demonstrated the bcr/abl gene, while 2 x 10(1) K562 cells among 2 x 10(6) total cells could be detected using the nested PCR method. In tests of seven clinical samples, five samples demonstrated bcr/abl and WT1 genes, while those in two other patients after bone marrow transplantation and a normal subject could not detected. This result suggests that our quantitative method reflect the clinical stages of CML patients.