Gain-of-function mutations in KIT a member of the receptor type tyrosine kinases are observed in certain neoplasms including mast cell tumors (MCTs) and acute myelogenous leukemias (AMLs). he receptor tyrosine kinase KIT regulates mast cell growth differentiation and survival1. When the KIT ligand stem cell factor (SCF) binds to this receptor KIT dimerizes autophosphorylates and consequentially activates various downstream signaling events including src family kinases (SFKs) phosphoinositide 3-kinase (PI3K). phospholipase Cγ (PLCγ) and mitogen-activated protein kinases (MAPKs)2. Specific gain-of-function mutations in KIT which induce constitutive autophosphorylation of KIT are observed in various tumors including gastrointestinal stromal tumors (GISTs)3 4 and mast cell tumors (MCTs)5 6 The V560G mutation in the juxtamembrane domain of KIT most frequently detected in GISTs is sensitive to tyrosine kinase inhibitors for example imatinib1 3 4 However most clinical cases of MCTs harbor the D816V mutation in the catalytic domain of KIT5 6 In contrast to GISTs MCTs expressing D816V KIT genes Sulindac (Clinoril) are imatinib-resistant7 8 Thus other approaches to control the mutated KIT-driven growth of MCTs are of clinical relevance9. We have reported that the activation Sulindac (Clinoril) of the inhibitory molecule CD72 could inhibit the growth of HMC1.2 cells a rapidly proliferating human mast cell line driven by V560G and D816V KIT10. CD72 is a transmembrane protein of the C type lectin family which contains ITIM motifs in its cytoplasmic domain11. The natural ligand for CD72 was identified to be CD100/Semaphorin 4D and specific antibodies against CD72 are known to mimic the consequences of ligation of CD72 by CD10012 13 Stimulation with CD100 or the agonistic antibodies to CD72 showed both positive and negative effects on B cell function which may be dependent on the stage of B cell development11. In human mast cells we observed that CD72 activation induced negative effects on mast cell growth and function10. As in B cells negative signals mediated via CD72 in mast cells are thought to be mediated by the formation of a CD72 – SHP-1 complex10 14 15 KIT mutations are also seen in patients with acute myeloid leukemias (AMLs) especially core binding factor AMLs (CBF-AMLs)16 17 18 CBF-AMLs are defined as AMLs with chromosomal aberrations affecting CBF transcription factor genes such as t(8; 21) and inv(16)19. The former aberrations produce the fusion protein AML1-ETO and the latter does another fusion protein CBFβ-MYH11. Studies using mouse models exposed that some mutations in tyrosine kinases are thought to be necessary for the development of CBF-AMLs in addition to the formations of the fusion proteins19. The Kasumi-1 cell collection provides a model for CBF-AML biology20. The Kasumi-1 cell collection which harbors an activating mutation in KIT was founded from a patient Sulindac (Clinoril) with AML M2 subtype21 22 and thus offered a model for CBF-AMLs16 17 18 In the case of the Kasumi-1 cell collection the N822K mutation in KIT is regarded as indispensable for the development22. Here we examined whether CD72 could inhibit the growth of Kasumi-1 cells as was observed with HMC1.2 cells. We display that Kasumi-1 cells communicate CD72 and that crosslinkage of CD72 suppressed the growth of the cells from the activation of SHP-1 and the producing down-regulation of SFKs and JNK. Therefore CD72 might present an opportunity for the targeted treatment of AMLs with KIT mutation. Results Kasumi-1 cells exhibit Compact disc72 To explore the ability of Compact disc72 to modify AML development we used the individual AML cell series Kasumi-1. We initial examined the expression of Compact disc72 proteins and mRNA in the cells. We performed RT-PCR to identify Compact disc72 mRNA using two pieces of primers concentrating on the exon1 to exon6 or exon2 to exon8 of Compact disc72 mRNA as previously defined10. The individual Burkitt’s lymphoma B cell series Raji was utilized being a Rabbit Polyclonal to MKNK2. positive control as well as the individual monocytic cell series U937 as a poor control23. The appearance of Compact disc72 mRNA was recognized in the Raji cells but not in the U937 cells once we expected (Fig. 1A). And we recognized the manifestation of CD72 mRNA in Kasumi-1 also (Fig. 1A). Next we executed western blotting analysis to confirm CD72 manifestation at protein level in the Kasumi-1 cells. This analysis showed the CD72 protein manifestation in the Sulindac (Clinoril) Kasumi-1 cells like Raji cells (Fig. 1B). Additionally we also carried out FACS analysis to confirm the surface manifestation of CD72 on the surface of Kasumi-1 cells using an anti-CD72 antibody BU40. This assay also exposed the manifestation of CD72 within the Kasumi-1 cells (Fig. 1C). Thus we determined the.