Previous work has demonstrated that WT1 (؊Ex5/؊KTS) potentiates granulocyte colony-stimulating factor (G-CSF)-mediated granulocytic differentiation. This WT1 isoform suppresses cyclin E, which may contribute to the prodifferentiation effect by slowing proliferation, but WT1 target genes that affect survival might also be involved. We screened a cDNA array and identified the bCL2 family member A1/BFL1 as a new WT1 target gene in 32D cl3 murine myeloblast cells. Induction of WT1 (؊Ex5/؊KTS) expression is accompanied by up-regulation of A1 on the cDNA array, and this up-regulation was confirmed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Moreover, both promoterreporter assays and chromatin immunoprecipitation assays suggest that this isoform of WT1 activates the promoter directly. Constitutive expression of A1 in 32D cl3 cells induces spontaneous granulocytic differentiation, with both morphologic and cell-surface antigen changes, as well as resistance both to chemotherapy and to withdrawal of interleukin-3 (IL-3). Finally, we note an association between WT1 expression and A1 expression in primary acute myeloid leukemia samples. Taken together, these results demonstrate that A1 is a new WT1 target gene involved in both granulocytic differentiation and resistance to cell death, and suggests that these genes might play an important role in the biology of high-risk leukemias.

WT1 was originally identified as a Wilms' tumor suppressor gene, but it may have oncogenic potential in leukemia and in some solid tumors. WT1 is a transcription factor that has been implicated in the regulation of target genes related to apoptosis, genitourinary differentiation, and cell cycle progression. Because induction of WT1 leads indirectly to increased p21 expression in osteosarcoma cells, we investigated the possibility that other genes involved in the G 1 /S phase transition might also be WT1 targets. Cyclin E plays a crucial role in the cell cycle by activating cyclin-dependent kinase 2, which phosphorylates Rb, leading to progression from G 1 into S phase. We identified several WT1 binding sites in the cyclin E promoter. We demonstrate that WT1 binds to these sites and that in transient transfection assays WT1 represses the cyclin E promoter. This activity is dependent on the presence of a binding site located downstream of the transcription start site. In intact cells, induction of WT1 expression down-regulates cyclin E protein levels. These results provide the first demonstration that WT1 can directly modulate the expression of a gene involved in cell cycle progression.WT1 was originally identified as a tumor suppressor gene in hereditary cases of Wilms' tumor. This gene encodes a 57-kDa protein with an amino-terminal transcriptional regulatory domain and a carboxyl-terminal zinc finger DNA binding domain (1, 2). WT1 mRNA is subject to two alternative splicing events leading to the generation of four distinct transcripts (3). The first alternative splice involves exon 5, which is either included in or excluded from the mature message. The other alternative splice involves a choice between two 3Ј splice acceptor sites at the beginning of exon 10. Selection of the more 5Ј splice acceptor site adds nine base pairs (referred to as the KTS insert for the 3 amino acids encoded by these base pairs) to exon 10. These 3 additional amino acids alter the spacing between the third and fourth zinc fingers, changing the DNA recognition site of the protein (4, 5). The subcellular localization of WT1 and its association with RNA splicing factors are also affected by the presence or absence of the KTS insert (6, 7).There are two classes of genes regulated by WT1. The first of these is composed of genes critical for the differentiation of the specific cell types that express WT1, in particular, the regulation of sex determination. For example, the Dax-1 promoter is dramatically up-regulated by the WT1 isoform lacking both exon 5 and the KTS insert (designated WT1(Ϫ/Ϫ)) and by the isoform that contains exon 5 but lacks the KTS insert (designated WT1(ϩ/Ϫ)), whereas the isoforms containing the KTS insert (designated WT1(Ϫ/ϩ)) and both exon 5 and the KTS insert (designated WT1(ϩ/ϩ)) have little effect (8). A similar pattern is seen in the regulation of the mullerian inhibitory substance and SRY promoters by WT1 (9, 10). Each of these target genes is important in differentiation of the genitourinary system.The second cl...

The activation threshold for antigen-specific T cell responses is dependent on the avidity of the trimolecular interaction between TCR, antigen, and MHC. We compared CD4+ T cell avidities for the diabetes-associated autoantigen glutamic acid decarboxylase 555-567 (GAD 555) among serial samples from autoantibody-positive subjects at high risk of progression to type 1 diabetes (T1D). T cells from three at-risk subjects demonstrated significant avidity increases (p<0.05 by F test) over time. This avidity shift correlated with the outgrowth of T cells expressing TCR BV 9, 15, 17 or 20 that demonstrated higher GAD 555 tetramer-binding levels compared to cells expressing other TCR BV genes. Similar analysis of autoantibody-negative, first-degree relatives and T1D patients did not demonstrate similar changes in avidity. These data implicate the outgrowth of T cells expressing higher affinity TCR in a process of antigen-specific T cell avidity maturation during the pre-clinical stage of T1D. KeywordsType 1 diabetes; CD4+ T cells; Autoimmunity; glutamic acid decarboxylase Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of insulin-producing beta cells of the pancreatic islets, resulting in hyperglycemia. At the time of diagnosis with T1D, approximately 20% of beta cells' insulin secretory capacity may remain [1]. The pre-clinical phase of T1D is marked by the presence of autoantibodies and frequently, T cells with specificity for beta cell antigens. In pre-diabetic patients, CD4+ T cell responses directed against pro-insulin and glutamic acid decarboxylase 555-567 (GAD 555) have been reported [2,3], and we observed that a cohort of autoantibody positive, at-risk subjects exhibited a significantly increased frequency of CD8+ T cells responding to an epitope of prepro-islet amyloid polypeptide [4]. However, T cells with the same specificities have also been isolated from healthy individuals, demonstrating that the mere presence of autoreactive T cells is not sufficient for diabetes induction [5,6]. Instead, it is more likely that the ability of a T cell population to induce and potentiate an autoaggressive response is dictated, in large part, by the threshold and magnitude of antigen responsiveness.Corresponding Author: Nathan E. Standifer, 1201 Ninth Ave., Seattle, WA 98101, Fax. (206) 223-7638, email: E-mail: NStand@benaroyaresearch.org. No potential conflicts of interest relevant to this article were reported.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Experimentally, thresholds for antigen response can be expressed in terms of T...