Kazushi Inoue

The WT1 gene encoding a zinc finger polypeptide is a tumor suppressor gene that plays a key role in the carcinogenesis of Wilms' tumor. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine relative levels of WT1 gene expression (defined in K562 cells as 1.00) in 45 patients with acute myelogenous leukemia (AML), 22 with acute lymphocytic leukemia (ALL), 6 with acute mixed lineage leukemia (AMLL), 23 with chronic myelogenous leukemia (CML), and 24 with non-Hodgkin's lymphoma. Significant levels of WT1 gene were expressed in all leukemia patients and for CML the levels increased as the clinical phase progressed. In striking contrast with acute leukemia, the levels of WT1 gene expression for NHL were significantly lower or even undetectable. Clear correlation was observed between the relative levels of WT1 gene expression (< 0.6 v > or = 0.6) and the prognosis for acute leukemia (AML, ALL, and AMLL). Patients with less than 0.6 levels had significantly higher rates of complete remission (CR), disease-free survival, and overall survival than those with > or = 0.6 levels, whereas CR could not be induced in any of the 7 patients with acute leukemia having greater than 1.0 levels of WT1 gene expression. The quantitation of the WT1 gene expression made it possible to detect minimal residual disease (MRD) in acute leukemia regardless of the presence or absence of tumor-specific DNA markers. Continuous monitoring of the WT1 mRNA was performed for 9 patients with acute leukemia. In 4 patients, MRD was detected 2 to 8 months before clinical relapse became apparent. In 2 other patients, the WT1 mRNA gradually increased after discontinuation of chemotherapy. No MRD was detected in the remaining 3 patients with AML who received intensive induction and consolidation therapy. Simultaneous monitoring of MRD by RT-PCR using primers for specific DNA markers in 3 patients (2 AML-M3 with PML/RAR alpha, and 1 AML-M2 with AML1/ETO) among these 9 patients detected MRD comparable with that obtained from quantitation of WT1 gene expression. In a patient with acute promyelocytic leukemia, the limits of leukemic cell detection by RT-PCR using either WT1 or promyelocytic leukemia/retinoic acid receptor-alpha gene primers were 10(-3) to 10(-4) and 10(-4) for bone marrow, and 10(-5) and 10(-4) for peripheral blood, respectively. Therefore, we conclude that WT1 is a new prognostic factor and a new marker for the detection of MRD in acute leukemia.

To clarify whether the expression of the WT1 gene in leukemic cells is aberrant or merely reflects that in normal counterparts, the expression levels of the WT1 gene were quantitated for normal hematopoietic progenitor cells. Bone marrow (BM) and umbilical cord blood (CB) cells were fluorescence-activated cell sorting (FACS)-sorted into CD34+ and CD34- cell populations, and the CD34+ cells into nine subsets (CD34+ CD33-, CD34+ CD33+, CD34+ CD38-, CD34+ CD38+, CD34+ HLA-DR-, CD34+ HLA-DR+, CD34+ c-kit(high), CD34+ c-kit(low), and CD34+ c-kit-) according to the expression levels of CD34, CD33, CD38, HLA-DR, and c-kit. Moreover, acute myeloid leukemic cells were also FACS-sorted into four populations (CD34+ CD33-, CD34+ CD33+, CD34- CD33+, and CD34- CD33-). FACS-sorted normal hematopoietic progenitor and leukemic cells and FACS-unsorted leukemic cells were examined for the WT1 expression by quantitative reverse transcriptase-polymerase chain reaction. The WT1 expression in the CD34+ and CD34- cell populations and in the nine CD34+ subsets of BM and CB was at either very low (1.0 to 2.4 x 10(-2)) or undetectable (< 10(-2)) levels (the WT1 expression level of K562 cells was defined as 1.0), whereas the average levels of WT1 expression in FACS-sorted and -unsorted leukemic cells were 2.4 to 9.3 x 10(-1). Thus, the WT1 expression levels in normal hematopoietic progenitor cells were at least 10 times less than those in leukemic cells. Therefore, we could not find any normal counterparts of BM or CB that expressed the WT1 at levels comparable with those in leukemic cells. These results indicate an aberrant overexpression of the WT1 gene in leukemic cells and imply the involvement of this gene in human leukemogenesis.

We have previously reported expression of WT1 in acute leukemia. To elucidate its biological significance, we examined the effect of the suppression of the WT1 expression by WT1 antisense oligomers on the growth of the leukemic cells expressing WT1. When 20 different WT1 antisense (AS) oligomers covering from the 5' cap sites of the WT1 gene to the 3' end were examined for the inhibitory effect on the growth of K562 cells expressing WT1, four WT1 AS oligomers inhibited the cell growth, whereas WT1 sense and random sequence oligomers had no effect on the cell growth of K562. Moreover, WT1 AS oligomers significantly inhibited the growth of the clonogenic cells of fresh leukemic cells in six of 14 patients with acute myeloid leukemia, in one of two patients with chronic myelogenous leukemia (CML) chronic phase, and in one of one patient with CML blastic crisis. However, these oligomers did not inhibit normal colony-forming unit-granulocyte-macrophage. Western blot analysis clearly demonstrated the significant reduction in the WT1 protein levels in the K562 and fresh leukemic cells that were treated with the WT1 AS oligomers, confirming that the inhibitory effect of the WT1 AS oligomers on the cell growth operates via the reduction in the WT1 protein levels. These results show that WT1 plays an important role in leukemogenesis.

The WT1 gene is a tumor-suppressor gene that was isolated as a gene responsible for Wilms' tumor, a childhood kidney neoplasm. We have previously reported that the WT1 gene is strongly expressed in leukemia cells with an increase in its expression levels at relapse and an inverse correlation between its expression levels and prognosis, thus making it a novel tumor marker for leukemic blast cells. Furthermore, WT1 antisense oligomers have been found to inhibit the growth of leukemic cells. These results strongly suggested the involvement of the WT1 gene in human leukemogenesis. The present study was performed to prove our hypothesis that the WT1 gene plays a key role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells, rather than a tumor-suppressor gene function. 32D cl3, an interleukin-3-dependent myeloid progenitor cell line, differentiates into mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF). However, when transfected wild-type WT1 gene was constitutively expressed in 32D cl3, the cells stopped differentiating and continued to proliferate in response to G-CSF. As for signal transduction mediated by G-CSF receptor (G-CSFR), Stat3alpha was constitutively activated in wild-type WT1-infected 32D cl3 in response to G-CSF, whereas, in WT1-uninfected 32D cl3, activation of Stat3alpha was only transient. However, most interesting was the fact that G-CSF stimulation resulted in constitutive activation of Stat3beta only in wild-type WT1-infected 32D cl3, but not in WT1-uninfected 32D cl3. Thus, WT1 expression constitutively activated both Stat3alpha and Stat3beta. A transient activation of Stat1 was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation, but no difference in its activation was found. No activation of MAP kinase was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation. These results demonstrated that WT1 expression competed with the differentiation-inducing signal mediated by G-CSFR and constitutively activated Stat3, resulting in the blocking of differentiation and subsequent proliferation. Therefore, the data presented here support our hypothesis that the WT1 gene plays an essential role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells and represent the first demonstration of an important role of the WT1 gene in signal transduction in hematopoietic progenitor cells.