Apoptotic function of an Ex4a(+)WT1 isoform.

<p>(<b>A</b>) The role of Ex4a(+)WT1 in apoptosis. Ex4a(+) or Mock vector was transfected into WT1-expressing HT-1080 cells. Frequencies (%) of Annexin V-positive apoptotic cells and cells with loss of MMP were determined by flowcytometry after 24 h. Left, Frequencies (%) of Annexin V-positive apoptotic cells are shown. Right, Frequencies (%) of cells with mitochondrial membrane potential (MMP) loss are shown. Results are means and S.D. of three independent experiments. *, p<0.05. (<b>B</b>) Expression of Ex4a(+) and major WT1 isoforms during apoptosis. K562 cells were treated with the indicated concentrations of Dox for 12 h and analyzed for Annexin-V positive apoptotic cells and expression of Ex4a(+) and major WT1 isoforms by flowcytometry and RT-PCR, respectively. Upper, Frequencies (%) of Annexin V-positive apoptotic cells. Lower, RT-PCR using Ex4-F and Ex6-R primer pair that amplifies both Ex4a(+) and major WT1 isoforms. GAPDH is used as an internal control. Results are representative of three independent experiments. (<b>C</b>) Change of Ex4a(+)WT1 and major WT1 isoforms during apoptosis. K562 cells were treated with the indicated concentrations of Dox for 12 h and expression of Ex4a(+)WT1 and total WT1 isoforms including both Ex4a(+) and major WT1 isoforms were determined by quantitative real-time RT-PCR using Ex4a-F and Ex6-R primer pair and Ex6-F and Ex7-R primer pair, respectively. Actin is used as an internal control for normalization. Expression levels of Ex4a(+)WT1 and total WT1 in Dox-untreated cells are defined as 1.0. (<b>D</b>) Suppression of Ex4a(+)WT1 inhibits Dox-induced apoptosis. K562 cells were transfected with one μg of either of two WT1 Ex4a-specific siRNAs (si-4a-1 and si-4a-2) or a control siRNA (si-control) together with 2.0 μg of Ex4a(+)WT1 vector or 2.0 μg of empty vector (Mock), cultured for 24 h, treated with 4.0 μM Dox for 12 h, and then analyzed for Annexin-V positive apoptotic cells by flowcytometry. Frequencies (%) of Annexin V-positive apoptotic cells are shown. Results are mean and S.D. of three independent experiments. *, p<0.05.</p

Isolation of a novel WT1 isoform.

<p>(<b>A</b>) Schematic representation of the human WT1 genomic structure (not to scale). Primers used for PCR amplification are indicated with arrows. Exons are shown as open boxes. Alternative splicing exon 5 (17AA) and KTS region in exon 9 are represented as black and hatched boxes, respectively. Translational start (ATG) and stop codons (TGA) are indicated. (<b>B</b>) Agarose gel electrophoresis of WT1 PCR products amplified using the primers indicated in (A) is shown. Bands were visualized by Gel-red staining. A novel transcript of 624 bp together with major WT1 isoforms, 17AA(+)WT1 (471 bp) and 17AA(-)WT1 (420 bp) were detected. Lane M, molecular marker (100 bp DNA ladder). (<b>C</b>) Upper, schematic representation of the WT1 genomic region encompassing exons 4 to 5, including the alternative splicing site in exon 4. Lower, schematic representation of the 17AA(+) and Ex4a(+)WT1 isoforms. Alternatively spliced exons 4a and 5 are shown in shaded and black boxes, respectively. DNA sequence of the newly identified 4a of 153 bp is expanded in the lower and surrounded by a solid line. Genomic sequences that follow immediately after the end of the 4a are shown in italic.</p

Ex4a(+)WT1 isoform produces C-terminal truncated WT1 proteins.

<p>(<b>A</b>) Schematic representation of 17AA(+)KTS(+)WT1 mRNA, structure of 17AA(+)KTS(+)WT1 protein, Ex4a(+)WT1 mRNA, and predicted structures of Ex4a(+)WT1 proteins. Translational start (ATG) and stop codons (TGA, TAA) are indicated. Four premature translational stop codons (two TAA and two TGA) are present in the reading frame in exon 4a. ZF represents zinc finger domain. (<b>B</b>) NMD inhibition by puromycin treatment. K562 cells were treated with 100 μg/ml of puromycin for 4 h to block NMD pathway and the amount of Ex4a(+)WT1 and MRP4 sv1-2 mRNA were determined by quantitative real-time PCR. The amount of each mRNA in puromycin-untreated K562 cells is defined as 1.0. Results are mean and S.D. of three independent experiments. *, p<0.05. (<b>C</b>) Stability of Ex4a(+)WT1 mRNA. K562 cells were treated with the transcription inhibitor ActD (5 μg/ml) and the amount of Ex4a(+)WT1 and MRP4 sv1-2 mRNA were determined by quantitative real-time PCR at indicated time points. The amount of Ex4a(+)WT1 and MRP4 sv1-2 mRNA were normalized to U6snRNA that were transcribed by RNA polymerase III and thus not blocked by ActD. The amount of each mRNA in ActD-untreated K562 cells (0 hours) is defined as 1.0. The trendlines are shown by black lines. (<b>D</b>) Schematic representation of three N-terminal His-tagged WT1 vectors. (<b>E</b>) Western blot analysis of total protein extracts from HT-1080 cells transfected with His-Mock, His-Ex4a(+)full, His-Ex1-4, or His-WTD expression vector. Transferred membrane was blotted with anti-His tag, 6F-H2 (specific for the N-terminal region of WT1 protein), or C-19 (specific for the C-terminal region of WT1 protein) antibody. MW represents molecular weight marker. Arrows indicate 30-KDa His-tagged truncated Ex1-4 WT1 protein. Results are representative of three independent experiments. (<b>F</b>) Endogenous Ex4a(+)WT1 mRNA is associated with polyribosomes. Polyribosomal fractions of K562 cells were purified by using the polyribosomal buffer with or without 100 mM EDTA (to release RNA from polyribosomes) and the amount of Ex4a(+)WT1 mRNA associated with polyribosomes was determined by quantitative real-time PCR using 4a-F and Ex6-R primer pair. The amount of Ex4a(+)WT1 mRNA in polyribosomes purified by using polyribosomal buffer without EDTA were defined as 1.0. Results are means and S.D. of three independent experiments. *, p<0.05.</p

Dominant negative function of an Ex4a(+)WT1 isoform.

<p>(<b>A</b>) Schematic representation of the EGFP reporter vector containing 400-bp Bcl-xL promoter (XL-400-EGFP), 1,480-bp Bcl-2 promoter (Bcl2-1480-EGFP), and the potential WT1 binding sites are shown. (<b>B</b>) HT-1080 cells were co-transfected by 0.5 μg of XL-400-EGFP or 0.5 μg of Bcl2-1480-EGFP vector together with 1.5 μg of empty vector (Mock), 1.5 μg of 17AA(+)KTS(-)WT1 (WTB), or 1.5 μg of WTB plus 1.5 μg of Ex4a(+)WT1 vector. A CMV promoter-driven DsRed expression vector (0.5 μg) was co-transfected with each sample to normalize for differences in transfection efficiency. Appropriate amounts of empty vector (Mock) were added to each transfection mixture to make a total of 4.0 μg of plasmid DNA. EGFP activities were measured by flowcytometry after 48 h transfection. Relative mean fluorescence intensity (MFI) of EGFP is shown. MFI in promoterless pEGFP1 plus Mock-transfected cells are defined as 1.0. Results are means and S.D. of three independent experiments. *, p<0.05. (<b>C</b>) Knockdown of Ex4a(+)WT1 isoform expression by siRNAs. K562 cells were transfected with either of two different Ex4a-specific siRNAs (si-4a-1 and si-4a-2) or control siRNA (si-control) for 48 h and Ex4a(+)WT1 mRNA expression was determined by quantitative real-time RT-PCR using 4a-F and Ex6-R primer pair. Actin is used as an internal control for normalization. Expression levels of Ex4a(+)WT1 in si-control-transfected cells are defined as 1.0. Results are means and S.D. of three independent experiments. *, p<0.05. (<b>D</b>) K562 cells were co-transfected by 5.0 μg of XL-400-EGFP or 5.0 μg of Bcl2-1480-EGFP reporter vector together with one μg of either of two WT1 Ex4a-specific siRNAs (si-4a-1 and si-4a-2) or control siRNA (si-control). A CMV promoter-driven DsRed expression vector (0.5 μg) was co-transfected with each sample to normalize for differences in transfection efficiency. EGFP activities were measured by flowcytometry 48 h after transfection. MFI of EGFP is shown. MFI in promoterless pEGFP1 vector plus si-control-transfected cells are defined as 1.0. Results are means and S.D. of three independent experiments. *, p<0.05. (<b>E</b>) K562 cells were transfected with either of two different Ex4a-specific siRNAs (si-4a-1 and si-4a-2) or control siRNA (si-control) for 48 h and Bcl-xL and Bcl-2 mRNA expression were determined by quantitative real-time RT-PCR. Actin is used as an internal control for normalization. Expression levels in Mock-transfected cells are defined as 1.0. Results are means and S.D. of three independent experiments. *, p<0.05.</p

Cloning and sequence analysis of full-length Ex4a(+) WT1 isoform.

<p>(<b>A</b>) 3’ rapid amplification of cDNA ends (3’ RACE) assay. Upper, 3’ RACE overview. cDNA was synthesized by using a 3’ RACE adaptor-dT primer. First PCR was performed by using exon 4a-specific forward and adaptor outer reverse primers. Second nested PCR was performed by using the first PCR product as a template with the nested Ex4a(+)WT1 cDNA-specific forward and the nested adaptor inner reverse primers. Lower, Agarose gel electrophoresis of the second nested PCR products is shown. Lane M, molecular marker (1 kbp DNA ladder). (<b>B</b>) Transcriptional start site of Ex4a(+)WT1. Upper, Schematic representation of the primer locations used to determine the transcriptional start site of Ex4a(+)WT1. Four (F1, F2, F3 and F4) and three (F5, F6, and F7) forward primers are located upstream and downstream, respectively, of the major transcriptional start site of WT1. Reverse primer (4a-R) is located in Exon 4a. The arrows indicate the primer positions used for PCR. +1 represents the major transcription start site of WT1. ATG indicates translational start codon. Lower, Agarose gel electrophoresis of PCR products amplified by using one each of 7 forward and 4a-R primers is shown. Lane M, molecular marker (1 kbp DNA ladder). (<b>C</b>) Alignment of nucleotide sequence of human 17AA(+)WT1 (upper lane) and Ex4a(+)WT1 (lower lane), and amino-acid (aa) sequences of exons 4 and 4a are shown. Asterisk indicates translational stop codon. Gaps are represented by dashes. The 4a sequence is marked in shaded grey. (<b>D</b>) Alignment of nucleotide sequence of the 4a and a part of intron 4 of WT1 in human, monkey, and mouse. The alignment is generated by CLUSTAL2.1 database with default parameters. The asterisks represent nucleotides identical to human and dashes represent an alignment gap. The 4a sequence of human WT1 is marked in shaded grey. Arrow indicates a novel alternative 5’ splice donor site. Genomic sequences of the exon 4a/intron 4 boundary region are highlighted in box.</p

Expression of Ex4a(+)WT1 isoform in human cancer cells.

<p>(<b>A</b>) Schematic representation of the WT1 exons and localization of the primers used for semi-quantitative RT-PCR (arrows) are shown. (<b>B</b>) Ex4a(+)WT1 mRNA expression was determined by RT–PCR using 4a-F and Ex6-R primer pair that amplifies only Ex4a(+)WT1 isoform in six different WT1-expressing cancer cells (AZ-521, HT-1080, LU99B, K562, Kasumi-1 and HL60) and one WT1-expressiong normal kidney cells 293. (<b>C</b>) Ex4a(+)WT1 mRNA expression in the paired samples of tumor (T) and normal tissues (N) of seven NSCLC was determined by RT-PCR as indicated in (B). (<b>D</b>) The ratio of Ex4a(+)WT1 to 17AA(+)WT1 isoforms was determined by RT–PCR using Ex4-F and Ex6-R primer pair that amplifies both Ex4a(+)WT1 and major WT1 isoforms in two different WT1-expressing cancer cells (LU99B and K562). (B-D) Actin is used as an internal control. Results are representative of three independent experiments.</p