All authors have read and approved the final manuscript.”
“Background NKG2D is a member of the NKG2 family of HLA class I C-type lectin receptors and is expressed as a homodimer by NK cells [1, 2] and cytotoxic lymphocytes [3, 4]. The ligands for NKG2D include the human class I-like

molecules MICA and MICB [5], which are stress-induced molecules expressed by tumors of epithelial origin [6, 7] and, leukemias [8], as well as by virus-infected cells [9, 10]. The recognition of the MICA and MICB ligands on tumor cells by the NKG2D receptor, found on NK cells, induces the cytotoxic activity of NK cells [11] and the subsequent selleck compound lysis of their tumor targets [12]. The secretion of MICA and MICB by cancer cells has been

suggested as a mechanism for tumor cell immune escape through the saturation of NKG2D receptors on cytotoxic cells [13, 14], thus abrogating their ability to recognize tumor cells. In fact, high levels of these molecules were BAY 80-6946 ic50 found in the sera of human cancer patients [15], and a direct correlation was found between increased serum concentrations of these molecules and tumor stage [16]. It is not known if the secretion of MICA and MICB by the tumor cells has any effect on the cancer cells themselves. This work was undertaken to determine if two human leukemic myelomonocytic cell lines, THP-1 and U-937, produce MICA and MICB and express NKG2D, and if these stress molecules induce cell proliferation. In order to determine if these properties are shared by other tumors, we also analyzed the CALO and INBL human epithelial cervical cancer cell lines. Methods Cells and antibodies The U-937 and THP-1 cell lines were purchased from ATCC (American Type Culture Collection), whereas CALO and INBL were established in our AZD6094 ic50 laboratory [17, 18]. The cells were cultured at 37°C with 5% CO2 in RPMI-1640 medium (Invitrogen) supplemented with 10% heat-inactivated FCS (Hyclone), 1-mM

MEM sodium Levetiracetam pyruvate solution, 2-mM MEM non-essential amino acids solution (Gibco), 0.1-mM L-glutamine, 100-U/ml penicillin and 100-μg/ml streptomycin (Gibco). Polyclonal antibody against MICA/MICB and murine monoclonal anti-MICA, anti-MICB and anti-NKG2D antibodies were purchased from R&D Systems. Proliferation assays U-937 and THP-1, as well as CALO and INBL, cells were plated at 5 × 103 cells per well in 96-well plates. Cells were treated with different concentrations of either MICA or MICB for 72 h at 37°C with 5% CO2 in RPMI-1640 containing 10% FCS. Proliferation was measured using the MTT assay (3-[4,5-Dimethylthiazol-2-4]-2,5-diphanyltetrazolium bromide) (Sigma). Briefly, 5 × 103 cells were cultured for 72 h in the presence of 1, 10, or 100 ng recombinant human MICA or MICB protein.