The c-myb proto-oncogene encodes the transcription factor, Myb, which is required for normal and malignant hematopoiesis. Aberrant expression and mutated forms of c-Myb have been associated with leukemias and solid tumors. Inhibition of c-myb gene expression in primary leukemic blast cells results in growth inhibition at doses where normal hematopoietic progenitor cells survive. One explanation for this differential sensitivity is that c-Myb regulates a unique set of genes in leukemic cells that are required for survival. To identify Myb gene targets in human leukemia cells, we utilized a microarray approach and K562 cells, engineered to express a tamoxifen inducible dominant negative Myb (MERT). When endogenous Myb activity was inhibited by tamoxifen in K562-MERT cells,104 genes changed in expression > 2-fold following microarray analysis. Of these, the gene encoding neuromedin U (NmU) exhibited the greatest change in expression [4.9-fold decrease]. NmU is expressed at moderate to high levels in tissues from the brain and gut as well as in monocytes and dendritic cells. Biologically active NmU, a 25-amino acid peptide, has been reported to stimulate smooth muscle contraction, increase blood pressure, alter ion transport in the gut, regulate local blood flow and modulate adrenocortical function. The function of NmU in hematopoietic cells has not been addressed. Consistent with our microarray data, quantitative real-time PCR (QRT-PCR) of tamoxifen treated K562-MERT cells revealed a 2.8-fold decrease in NmU expression compared to untreated cells. In primary acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) cells, c-myb expression was elevated, while only the AML cells had elevated NmU expression compared to normal peripheral blood mononuclear cells as determined by QRT-PCR. To investigate the function of NmU in hematopoietic cells, we examined the effect of NmU on colony growth of K562-MERT cells in methylcellulose. Exogenous NmU “rescued” growth suppression in K562-MERT cells indicating that NmU stimulates proliferation of myeloid leukemia cells. Western blot analyses revealed the presence of NmU receptor type-1 (NMU1R) in K562 cells and primary AML cells. Finally, we determined the ability of NmU to induce calcium ion (Ca+2) flux in K562 cells, because signaling through NMU1R has been shown to induce an intracellular Ca+2 flux in Cos-7, HEK-293, and CHO cells. Within 100 seconds of adding NmU to Indo-1 labeled K562 cells, we observed an accumulation of intracellular Ca+2. Taken together, these results suggest that NmU expression is regulated, at least in part, by c-myb, that NmU/NMU1R constitutes an autocrine loop that plays a role in proliferation of myeloid leukemia cells, and that NmU-mediated proliferation involves intracellular Ca+2 flux. The exact nature, and importance, of NmU function in malignant hematopoietic cells is currently under investigation in our laboratory.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]