Cb2 is a novel protooncogene encoding the peripheral cannabinoid receptor. Previous studies demonstrated that 2 distinct noncoding first exons exist: exon-1A and exon-1B, which both splice to proteincoding exon-2. We demonstrate that in retrovirally induced murine myeloid leukemia cells with proviral insertion in Cb2, exon-1B/exon-2 Cb2 messenger RNA levels have been increased, resulting in high receptor numbers. In myeloid leukemia cells without virus insertion in this locus, low levels of only exon-1A/exon-2 Cb2 transcripts were present and receptors could not be detected. To elucidate the function of Cb2 in myeloid leukemia cells, a set of in vitro experiments was carried out using 32D/G-CSF-R (granulocyte colony-stimulating factor receptor) cells transfected with exon-1B/exon-2 Cb2 complementary DNA and a myeloid cell line carrying a virus insertion in Cb2 (ie, NFS 78). We demonstrate that a major function of the Cb2 receptor is stimulation of migration as determined in a transwell assay. The cannabinoid receptors belong to the superfamily of 7-transmembrane G protein-coupled receptors (GPCRs). Several GPCRs have been shown to be involved in cell growth and oncogenesis as the result of aberrant expression. [5][6][7] Examples of GPCRs with transforming ability are the ␣1B-adrenergic, 8 thrombin, 9 and serotonin 1c receptors 10 and the receptor encoded by MAS oncogene. 11,12 Our previous observation that Cb2 is a common virus integration site suggests that aberrant expression of this 7-transmembrane receptor may be a critical event in transformation in certain cases of leukemia. 3 The protein-coding region of Cb2 is located on a single exon (exon-2) of approximately 4 kilobases. Recently we identified 2 distinct 5Ј noncoding exons (ie, exon-1A and exon-1B) previously designated exon-1 and exon-1Ј, respectively. 3 In the study presented here we first carried out experiments to investigate Cb2 messenger RNA (mRNA) transcripts and protein expression in leukemic cells with or without retroviral insertion in the Cb2 locus. Secondly, we performed studies to determine the function of the peripheral cannabinoid receptor when overexpressed in myeloid cells.GPCRs have been related to many functions, including cell proliferation, maturation, survival, apoptosis, or migration. 6,13,14 In the present study, we investigated the function of the peripheral cannabinoid receptor when overexpressed on myeloid cells (ie, 32D/G-CSF-R [granulocyte colony-stimulating factor receptor]) in which we overexpressed exon-1B/exon-2 Cb2 splice variant and a myeloid leukemia cell line containing a virus insertion in the Cb2 locus, NFS 78. We also wished to determine which of the large panel of Cb2 ligands that have been identified previously is the true agonist of the receptor. We investigated the effects of natural (␦ 8 24 ) cannabinoids. We show that 2-AG is the most potent agonist for the Cb2 receptor and that a major function of 2-AG is stimulation of migration. We further studied whether 2-AG acts as a chemotactic or chemokinetic age...
The leukemia and lymphoma disease locus Evi12 was mapped to the noncoding region of a novel gene, Gnn (named for Grp94 neighboring nucleotidase), that is located immediately upstream of the Grp94/Tra1 gene on mouse chromosome 10. The Gnn gene is conserved in mice and humans. Expression of fusion constructs between GFP and Gnn cDNA isoforms in HEK-293 cells showed that Gnn proteins are located mainly in the cytoplasm. Immunoblotting experiments demonstrated the presence of multiple Gnn protein isoforms in most organs, with the lowest levels of expression of the protein detected in bone marrow and spleen. The Evi12-containing leukemia cell line NFS107 showed high levels of expression of a ϳ150-kDa Gnn isoform (Gnn 107 ) that was not observed in control cell lines. Overexpression may be due to the viral insertion in Evi12. The Gnn 107 protein is probably encoded by a Gnn cDNA isoform that is expressed exclusively in NFS107 cells and that includes sequences of TU12B1-TY, a putative protein with homology to 5-nucleotidase enzymes. Interestingly, using Affymetrix gene expression data of a cohort of 285 patients with acute myeloid leukemia (AML), we found that GNN/TU12B1-TY expression was specifically increased in two AML clusters. One cluster consisted of all AML patients with a t(8;21) translocation, and the second cluster consisted of AML patients with a normal karyotype carrying a FLT3 internal tandem duplication. These findings suggest that we identified a novel proto-oncogene that may be causally linked to certain types of human leukemia.
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