Constitutive expression of the Wilms’ tumor gene WT1 inhibits the differentiation of myeloid progenitor cells but promotes their proliferation in response to granulocyte-colony stimulating factor (G-CSF)
Introduction
The Wilms’ tumor gene WT1 was identified as a gene responsible for Wilms’ tumor, a childhood kidney neoplasm and classified as a tumor suppressor gene [1], [2]. The WT1 gene encoding a zinc finger transcription factor represses transcription of growth factors [3], [4], [5], growth factor receptor [6] and the other genes [7], [8].
We [9], [10], [11] and others [12], [13], [14], [15] have reported high levels of wild-type WT1 expression in fresh leukemia cells regardless of the types of leukemias (acute myeloid leukemia [AML], acute lymphoid leukemia [ALL], and chronic myeloid leukemia [CML]). An inverse correlation between WT1 expression levels and prognosis was found [9], and WT1 expression levels significantly increased after relapse of acute leukemia [16]. Furthermore, WT1 antisense oligomer specifically inhibited the growth of leukemia cells [17]. In a recent report of ours [18], we showed that WT1-non-expressing 32D cl3 (an IL-3-dependent myeloid progenitor cell line) could completely differentiate into mature neutrophils by the stimulation with granulocyte-colony stimulating factor (G-CSF), whereas constitutively WT1-expressing 32D cl3 stopped differentiating, but instead continued to proliferate in response to G-CSF. Our serial studies [9], [10], [11], [16], [17], [18] strongly suggested that WT1 plays an essential role in human leukemogenesis and may function as an oncogene rather than as a tumor suppressor gene in hematopoietic progenitor cells.
The majority of AML cells characterized by maturation arrest within the myeloid lineage proliferate dependently on growth factors like their normal counterparts [19], but are refractory to differentiation induction [20]. Most responsive AML cells proliferate, but do not differentiate in response to G-CSF stimulation [21], suggesting that the G-CSF signaling pathway is altered in AML cells.
The WT1 gene is also expressed in normal hematopoietic progenitor cells, and the expression is downregulated along with the differentiation of the hematopoietic progenitor cells [9], [11], [22], [23], [24], [25]. Thus, the WT1 gene is thought to play an important role in proliferation and differentiation of normal hematopoietic progenitor cells.
The aim of the present study was to prove our hypothesis that the WT1 gene showing strong expression in almost all leukemia cells performs an oncogenic function in hematopoietic progenitor cells and thus promotes their proliferation, but inhibits their differentiation in response to G-CSF through the alteration of G-CSF signaling pathway. We describe here that constitutive expression of the WT1 gene in hematopoietic progenitor cells promotes their proliferation, but instead inhibits their differentiation in response to G-CSF.
Section snippets
Construction of retroviral vectors
Murine retroviral vectors capable of expressing the full-sized, non-spliced form (i.e. 17 amino acids (+), KTS (+)) of human WT1 were constructed with the pM5Gneo retroviral vector as described previously [18]. This vector derived from murine proliferative sarcoma virus contains viral long terminal repeats (LTRs) and a neomycin resistance gene (NeoR) under the control of herpes virus thymidine kinase promoter.
Infection of bone marrow cells with WT1-containing recombinant retrovirus
The pM5Gneo retroviral vector with or without the WT1 gene was transfected into a
Increase in the number of myeloid colonies in response to G-CSF by constitutive expression of the WT1 gene
Bone marrow (BM) cells that were concentrated for hematopoietic progenitor cells by in vivo treatment with 5-FU were infected with a recombinant retrovirus containing a neomycin resistance gene plus a human full-sized, non-spliced type WT1 cDNA (Fig. 1) in the presence of rmSCF (100 ng/ml), rmIL-3 (10 ng/ml), and rhIL-6 (10 ng/ml). The BM cells were cultured with the addition of G418 (1.0 mg/ml) for a further 3 days in order to select WT1 (Neo)-infected BM cells. The selected BM cells were then
Discussion
G-CSF gives two signals, proliferation and differentiation-inducing signals, to myeloid progenitor cells through G-CSF receptor (G-CSFR). The G-CSFR consists of two distinct functional domains. The membrane-proximal region containing two subdomains (boxes 1 and 2) transduces the proliferation signal and the C-terminal domain (box 3) transduces the differentiation-inducing signal [27], [28], [29]. Our present study showed that constitutive WT1 expression in myeloid progenitor cells by the
Acknowledgements
We thank Tsuyomi Yajima for preparation of the manuscript and Machiko Mishima for her skillful technical assistance.
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