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The c-myb proto-oncogene has been implicated in leukemogenesis, but possible mechanisms remain ill defined. To gain further insight to this process, we used transcript profiling in K562 cells expressing a dominant-negative Myb (MERT) protein. A total of 105 potential Myb gene targets were identified. Neuromedin U (NmU), a peptide affecting calcium transport, underwent the greatest expression change (ϳ 5-fold decrease). To verify a linkage between c-myb and NmU, their mRNA levels were quantitated using realtime polymerase chain reaction in primary acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), as well as normal hematopoietic cells. We found that c-myb was elevated in AML and ALL samples, but NmU expression was increased only in AML cells. Significantly, only AML cells expressed the cognate receptor of NmU, NMU1R, suggesting the presence of a novel autocrine loop. We examined this possibility in detail. Exogenous NmU "rescued" growth suppression in K562-MERT cells and stimulated the growth of primary AML cells. Short interfering RNA "knockdown" of NmU in K562 cells arrested cell growth. Exposing Indo-1-labeled K562 cells to NmU induced an intracellular Ca ؉؉ flux consistent with engagement of the NMU1R. Combined, these results suggest that NmU expression is related to Myb and that the NmU/NMU1R axis constitutes a previously unknown growth-promoting autocrine loop in myeloid leukemia cells. IntroductionThe proto-oncogene c-myb encodes a transcription factor, Myb, that is expressed predominantly in immature hematopoietic cells [1][2][3] where it plays a critical role in definitive hematopoiesis. 4-6 c-Myb transactivates its target genes by binding to a well-defined consensus sequence, [PyAAC(T/G)G], referred to as the Myb responsive element. 7 Of genes known to be induced by Myb, many regulate cell proliferation, differentiation, and survival including c-myc, [8][9][10][11][12] cdc2, 13 c-kit, [14][15][16] 17 mim1, 18 myeloblastin, 19 and bcl2. 20,21 The oncogenic potential of myb was first observed in birds, when it was discovered that a truncated form of the cellular proto-oncogene, called viral-myb (v-myb), was contained within the AMV and E26 leukemia viruses. [22][23][24] v-myb has deletions at its 5Ј and 3Ј ends, which results in a protein missing a small, but apparently inconsequential, portion of its DNA-binding domain and virtually its entire negative regulatory domain. Loss of the negative regulatory domain is thought to contribute to the transforming ability of the protein. 25 These deletions may have arisen as a result of retroviral insertional mutagenesis because the myb locus is known to undergo retroviral insertion. 22 Further, retroviral insertion of c-myb in chicken and mouse models produces a truncated form of the c-myb gene, which causes B-and T-cell lymphomas [26][27][28][29] and myeloid leukemias, 10,30-32 respectively.In humans, aberrant expression of c-myb has been associated with leukemia 33 as well as a number of solid tumor cancers such as colon cancer. 34 Exposing human c...

The c-Myb and GATA-3 transcription factors play important roles in T-cell development. We recently reported that c-Myb, GATA-3, and Menin form a core transcription complex that regulates GATA-3 expression and ultimately Th2 cell development in human peripheral blood T-cells. However, c-Myb roles for Th2 cytokine expression were not demonstrated. Here, we report that c-Myb and GATA-3 cooperatively play an essential role in IL-13 expression though direct binding to a conserved GATA-3 response element (CGRE), an enhancer for IL-13 expression. c-Myb and GATA-3 were shown to activate the CGRE-IL-13 promoter by ~160 fold, and mutation of the canonical Myb binding site completely abrogated CGRE enhancer activity. In contrast, mutation of the GATA binding site partially decreased CGRE enhancer activity. GATA-3 did not bind to CGRE when c-myb expression was silenced. c-Myb, GATA-3, Menin and MLL bound to CGRE in human primary CD4+ effector/memory cells. Moreover, c-myb silencing significantly decreased both methylation of histone H3K4 and acetylation of histone H3K9 at the IL-13 locus in CD4+ effector/memory cells. Therefore, in addition to the strong enhancer effect for the transcription of IL-13, the c-Myb/GATA-3 complex recruits MLL to the CGRE for histone modification of the IL-13 locus during the differentiation of memory Th2 cells.

A 2',5'-oligoadenylate (2-5A)-dependent 37-kDa form of RNase L has been reported in extracts of peripheral blood mononuclear cells (PBMC) from individuals with chronic fatigue syndrome (CFS). In the current study, analytic gel permeation FPLC, azido photoaffinity labeling, two-dimensional (2-D) gel electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been used to examine the biochemical relationship between the 80-kDa RNase L in healthy control PBMC and the 37-kDa RNase L in PBMC from individuals with CFS. Like the 80-kDa RNase L, the 37-kDa RNase L is present as a catalytically inactive heterodimer complex with the RNase L inhibitor (RLI). Formation of a 37-kDa RNase L-RLI complex indicates that the 37-kDa RNase L is structurally similar to the 80-kDa RNase L at the N-terminus, which contains the 2-5A binding domain. The enzymatically active monomer form of 37-kDa RNase L resolved by 2-D gel electrophoresis has a pI of 6.1. RT-PCR and Southern blot analyses demonstrated that the 37-kDa RNase L is not formed by alternative splicing. In-gel tryptic digestion of the 37-kDa RNase L that was excised from 2-D gels and subsequent MALDI-MS analysis identified three peptide masses that are identical to three predicted peptide masses in the 80-kDa RNase L. The electrophoretic mobility of 2-5A azido photolabeled/immunoprecipitated 37-kDa RNase L was the same under reducing and nonreducing conditions. The results presented show that the 37-kDa form of RNase L in PBMC shares structural and functional features with the native 80-kDa RNase L, in particular in the 2-5A binding and catalytic domains.