Cells were infected with an (contamination

Cells were infected with an (contamination. the EPIYA-C or EPIYA-D motif (17). c-Src is usually then subsequently dephosphorylated and inactivated by a negative feedback loop brought on by the binding of PDGFD phosphorylated CagA (p-CagA) to the C-terminal Src kinase (CSK) (18, 19). The tyrosine kinase c-Abl maintains EPIYA-A, EPIYA-B, and EPIYA-C/D phosphorylation of CagA at later time points at one or two sites (17). In the cytoplasm, translocated CagA can interact with several intracellular signaling proteins in a phosphorylation-dependent as well as phosphorylation-independent manner (20). As a consequence, CagA-mediated deregulation of downstream signaling pathways induces a drastic Src Inhibitor 1 epithelial cell elongation (21,C23). Based on the knowledge that prolonged bacterial colonization prospects to the infiltration of neutrophils and lymphocytes into the mucosal epithelium (2, 24), it was hypothesized that can directly interact with cells of the immune system. However, compared to information about gastric epithelial cells, the understanding of CagA functions in nonepithelial cells is rather limited. Previous studies were conducted in different types of professional phagocytes of the monocytic lineage, including THP-1, U937, J774A.1, and Josk-M. In these cell types, efficient T4SS-dependent CagA translocation and tyrosine phosphorylation have been exhibited (25, 26). Further, a tyrosine-phosphorylated C-terminal CagA fragment was recognized, indicating that CagA is usually rapidly cleaved into an N-terminal fragment exhibiting a molecular mass of approximately 100 kDa and a C-terminal part with a molecular mass of approximately 40 kDa with unknown functions (25, 26). The high incidence of MALT lymphoma in Src Inhibitor 1 prolonged infections suggests that B cells might be directly infected by as well. Recently, CagA translocation and tyrosine phosphorylation were observed in the B cell collection BJAB (27). In B lymphocytes, CagA was shown to interact with the Src homology 2 domain name tyrosine phosphatase (SHP-2) leading to the induction of mitogen-activated protein kinases and upregulation of the antiapoptotic proteins Bcl-2 (B cell lymphoma 2) and Bcl-X (27). Although these data Src Inhibitor 1 show a possible contribution of CagA to the formation of MALT lymphoma, the signaling events leading to CagA tyrosine phosphorylation remained unclear. In this study, we investigated CagA translocation and tyrosine phosphorylation in the B cell collection MEC1, which is derived from a B cell chronic lymphocytic leukemia (B-CLL) patient (28). The nonreceptor tyrosine kinases Src and c-Abl functioned as potent CagA kinases in B Src Inhibitor 1 cells, mediating phosphorylation Src Inhibitor 1 of the EPIYA motifs in CagA. Tyrosine phosphorylation of CagA could efficiently be blocked by the Src and Abl inhibitor dasatinib, and thus Src and Abl represent possible targets in the treatment of CagA-positive MALT lymphoma. MATERIALS AND METHODS Cell culture and inhibitor treatment. AGS, MEC1, and U937 cells were cultured in RPMI 1640 medium (Sigma, Germany) supplemented with 2 mM l-glutamine (Biowest, Germany) and 10% fetal calf serum (FCS) (Biowest, France) in a humidified 5% CO2 atmosphere at 37C (Table 1). Adherent AGS cells were seeded in tissue culture dishes 48 h before contamination and produced to 70% confluence. At 24 h prior to contamination, medium was replaced by new serum-free medium. Suspension cells (MEC1 and U937) were harvested by centrifugation at 250 at 4C for 5 min, and 5 106 cells were seeded in 100-mm tissue culture dishes with serum-free medium at 24 h prior to contamination. Where indicated, cells were pretreated with 10 M PP2 to block Src kinases (Calbiochem, Austria), imatinib mesylate (STI-571; Gleevec) to block c-Abl, or dasatinib to block Src and Abl kinases (LC Laboratories, MA) for 30 min prior.