The peripheral cannabinoid receptor Cb2 in leukemia

Acute myeloid leukemia (AML) is a blood cell disorder characterized by an accumulation of immature blasts in bone marrow and blood. Human AML is frequently characterized by non-random chromosome translocations resulting in the generation of specific transforming fusion genes and fusion proteins, of which a significant number has been cloned, e.g. AML1-ETO fusion gene in AML with a t(8;21) translocation or PML-RAR in cases with translocation t(15;17). However, in approximately 40 - 50% of AML cases no chromosomal abnormalities are evident, indicating that other more subtle mutations are responsible for the leukemic transformation of myeloid precursor cells. Moreover, AML, like other cancers, is a multigenic disease resulting from an accumulation of multiple genetic aberrations. Thus even in cases with well-characterized translocations, additional genetic defects have likely contributed to the development of AML. The identification and functional analysis of novel disease genes in AML is a major goal of our research group. One approach utilized to identify novel disease genes in leukemia is retroviral insertional mutagenesis. Mice injected with murine leukemia viruses (MuLVs) develop leukemia following proviral insertion into or near potential disease genes. Viral insertions found in a particular locus in independent tumors are called common virus integration sites, cVIS, and mark the locations of potential proto-oncogenes or tumor suppressor genes. The mouse strain and the type of retrovirus used will determine the kind of leukemia that will develop. We used NIH/Swiss mice injected with Cas-Br-M MuLV which develop frequently myeloid leukemias. Using this combination, we previously identified the cVIS Evi11 and demonstrated that the gene encoding the peripheral cannabinoid receptor Cb2 is the likely target gene. Cb2 encodes a seven transmembrane receptor that belongs to the G proteincoupled receptor (GPCR) family and is predominantly present on B lymphocytes. The main objective of the work presented in this thesis is to determine whether Cb2 is indeed a proto-oncogene and, if so, by which mechanism it may transform hematopoietic precursor cells

Hematopoietic cells expressing the peripheral cannabinoid receptor migrate in response to the endocannabinoid 2-arachidonoylglycerol

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 protein-coding 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. Exposure of Cb2-expressing cells to different cannabinoids showed that the true ligand for Cb2 is 2-arachidonoylglycerol (2-AG), which may act as chemoattractant and as a chemokinetic agent. Furthermore, we observed a significant synergistic activity between 2-AG and interleukin-3 or G-CSF, suggesting cross-talk between the different receptor systems. Radioactive-ligand binding studies revealed significant numbers of Cb2 receptors in normal spleen. Transwell experiments carried out with normal mouse spleen cells showed 2-AG-induced migration of B220-, CD19-, immunoglobulin M-, and immunoglobulin D-expressing B lymphocytes. Our study demonstrates that a major function of Cb2 receptor expressed on myeloid leukemia cells or normal splenocytes is stimulation of migration

Paranthidium, Anthidiellum, and Hypanthidium

9 p. ; 24 cm.Includes bibliographical references

Toll-like receptor 2 expression on c-kit + cells tracks the emergence of embryonic definitive hematopoietic progenitors

International audienceHematopoiesis in mammalian embryos proceeds through three successive waves of hema-topoietic progenitors. Since their emergence spatially and temporally overlap and phenotypic markers are often shared, the specifics regarding their origin, development, lineage restriction and mutual relationships have not been fully determined. The identification of wave-specific markers would aid to resolve these uncertainties. Here, we show that toll-like receptors (TLRs) are expressed during early mouse embryogenesis. We provide phenotypic and functional evidence that the expression of TLR2 on E7.5 c-kit + cells marks the emergence of precursors of erythro-myeloid progenitors (EMPs) and provides resolution for separate tracking of EMPs from primitive progenitors. Using in vivo fate mapping, we show that at E8.5 the Tlr2 locus is already active in emerging EMPs and in progenitors of adult hematopoietic stem cells (HSC). Together, this data demonstrates that the activation of the Tlr2 locus tracks the earliest events in the process of EMP and HSC specification

DNMT1-interacting RNAs block gene specific DNA methylation

Summary DNA methylation was described almost a century ago. However, the rules governing its establishment and maintenance remain elusive. Here, we present data demonstrating that active transcription regulates levels of genomic methylation. We identified a novel RNA arising from the CEBPA gene locus critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extended the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene selective demethylation of therapeutic targets in disease