Real-time quantitative reverse transcriptase polymerase chain reaction method was established for detecting the expression levels of WT1 gene and WT1+17AA isoforms in 226 acute myeloid leukemia (AML) bone marrow (BM) cells. The results showed that WT1 gene was 2-3 logarithms expressed more in AML BM cells at initial diagnosis or relapse than in normal BM cells (p<0.001), with predominant WT1+17AA isoforms expression (the ratio of WT1+17AA/WT1 more than 0.50). Interestingly the ratio of WT1+17AA/WT1 was statistically higher in relapsed AMLs than in initially diagnosed (p=0.01), speculating that WT1+17AA isoforms might participate in AML relapse.
"All of the four isoforms are expressed in primary human solid cancers (Oji et al., 2002) and human primary leukemia (Miwa et al., 1992). Among the four WT1 isoforms, the WT1 17AA(+)KTS(+) isoform is dominantly expressed in all of the above mentioned cancers (Haber et al., 1991; Gu et al., 2010). This suggests that the constitutive expression of the WT1 17AA(+)KTS(+) isoform could rescue the growth inhibitory effect of WT1 antisense oligomers in solid tumors (Oji et al., 1999) indicating the contribution of the WT1 17AA(+)KTS(+) isoform to the growth of cancer cells (Hubinger et al., 2001). "
[Show abstract][Hide abstract] ABSTRACT: Wilms' tumor (WT1) gene overexpresses in leukemic cells which is alternatively spliced at two sites, yielding four isoforms: WT1 (+/+), (+/-), (-/+), and (-/-). Curcumin is one of major active components of the spice turmeric, widely known as anticancer. This study investigated the effects and inhibitory mechanism of pure curcumin on WT1 isoform-transfected U937 cells. WT1 transfected U937 cells were initially treated for 24 h, with 10 µM pure curcumin. Pure curcumin exhibited a strong inhibitory effect on WT1 (+/+) mRNA level detected by real time PCR. Treatment of WT1 transfected U937 cells with non-cytotoxic doses (10, 15, and 17 µM) of pure curcumin decreased WT1 protein levels in a dose-dependent manner. Pure curcumin at the concentration of 15 µM significantly decreased the protein levels of the WT (+/+) isoform in a time-dependent manner. It also decreased exogenous WT1 (+/+) protein half-life. WT1 protein expression was inhibited by protein kinase C inhibitor (GF109203x) suggesting that pure curcumin decreased exogenous WT1 (+/+) expression in transfected U937 cells via protein kinase C during post-translational processing.
[Show abstract][Hide abstract] ABSTRACT: Wilms' tumour is a paediatric malignancy of the kidneys and is one of the most common solid childhood cancers. The Wilms' tumour 1 protein (WT1) is a transcription factor that can either activate or repress genes involved in growth, apoptosis and differentiation. It is frequently mutated or aberrantly expressed in Wilms' tumour, where the wild type protein would normally act as a tumour suppressor. Several studies, however, have found that wild type WT1 acts as an oncogene in adult tumours, primarily through the inhibition of apoptosis. The expression of WT1 correlates with the aggressiveness of several adult cancers, and its continued expression following treatment is indicative of a poor outcome.We recently found that the treatment of tumour cell lines with cytotoxic drugs leads to the cleavage of WT1 by the serine protease HtrA2. HtrA2 binds to a specific region of WT1, the suppression domain, and then cleaves WT1 at multiple sites. The HtrA2-mediated proteolysis of WT1 leads to its removal from gene promoter regions and changes in gene expression. Cleavage of WT1 by HtrA2 enhances apoptosis. This event is advantageous to the treatment of adult tumours where WT1 acts as an oncogene. However, when WT1 is acting as a tumour suppressor in paediatric malignancies, proteolysis by HtrA2 would be antagonistic to therapy.
[Show abstract][Hide abstract] ABSTRACT: Wilms' tumor gene 1 (WT1) functions including some contradictory effects may be explained by the presence and interactions of its isoforms, however, their evaluation has been so far complicated by several technical problems. We designed unique quantitative PCR systems for direct quantification of the major WT1 isoforms A[EX5-/KTS-], B[+/-], C[-/+] and D[+/+] and verified their sensitivity, specificity and reproducibility in extensive testing. With this method we evaluated WT1 total and isoform expression in 23 normal bone marrow (BM) samples, 73 childhood acute myeloid leukemia (AML), 20 childhood myelodysplastic syndrome (MDS), 9 childhood severe aplastic anemia (SAA), 30 adult AML and 29 adult MDS patients. WT1 isoform patterns showed differences among these samples and clustered them into groups representing the specific diagnoses (P<0.0001). Isoform profiles were independent of total WT1 expression and possess certain common features-overexpression of isoform D and EX5[+] variants. The KTS[+]/KTS[-] ratio was less variable than the EX5[+]/EX5[-] ratio and differed between children and adults (P<0.001); the EX5[+]/EX5[-] ratio varied between diagnoses (AML vs MDS, P<0.001). These findings bring new insights into WT1 isoform function and suggest that the ratio of WT1 isoforms, particularly EX5 variants, is probably crucial for the process of malignant transformation.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 03/2012; 26(9):2086-95. DOI:10.1038/leu.2012.76 · 10.43 Impact Factor
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