Blocking CCR7 with a monoclonal antibody resulted in a decreased homing capacity of ALL cells histology (Figures 1 and ?and2)2) one-sided screening was chosen, since death rates at a time point in the control groups were known and we aimed to detect a difference in one direction. increased risk of CNS relapses in BCP-ALL,7 and we recently recognized the MER tyrosine kinase as a marker for CNS involvement in that entity.8 Most chemotherapeutic drugs applied for ALL treatment poorly penetrate the blood-brain barrier. Accordingly, current pediatric treatment protocols advocate considerable intrathecal and systemic chemotherapy,9C11 which has been associated with long-term neurologic sequelae.11,12 Thus, more precise diagnostic and prognostic markers for CNS involvement in ALL are needed, not only to control CNS infiltration but also to avoid systemic overtreatment. Zeta-chain-associated protein kinase 70 (ZAP70), a tyrosine kinase mainly expressed in T cells and natural killer Rabbit Polyclonal to OR4L1 (NK) cells, is also found to be expressed at low levels in B cells. 13 The role of ZAP70 in BCP-ALL is usually poorly explained. Few reports have shown that ZAP70 is usually expressed in some ALL cell lines and in a report of 5 patients with the fusion.14C16 ZAP70 has been shown to be overexpressed in B-cell chronic lymphocytic leukemia (B-CLL), indicating an aggressive course of the disease.17 Clemizole hydrochloride ZAP70 expression and phosphorylation have been associated with B-cell Clemizole hydrochloride receptor (BCR) signaling in B-CLL.18,19 Further, ZAP70 may function as an adaptor protein and enhance BCR signaling independently of its kinase activity.20 ZAP70 has been shown to enhance the migration of malignant B cells to the bone marrow (BM) by upregulating adhesion molecules and chemokine receptors (CCRs),21,22 and is also required for C-X-C motif chemokine ligand 12 (CXCL12)-mediated T-cell transendothelial migration.23,24 In T-cell acute lymphoblastic leukemia (T-ALL), the chemokine receptors CCR7 and CXCR4 have been associated with an increased capability of T-ALL cells to enter the CNS, mainly in cell collection models.25,26 We hypothesized that ZAP70 mediates the infiltration and the survival of ALL cells in the CNS. Downregulating ZAP70 in ALL cell lines resulted in a reduced CCR7/CXCR4 expression and an impaired CNS infiltration in NSG mice the regulation of ERK. In contrast, up-regulating ZAP70 caused an enhanced CCR7/CXCR4 expression and an improved migratory capacity towards chemokine ligand 19 (CCL19) and CXCL12 gradients. Blocking CCR7 with a monoclonal antibody resulted in a decreased homing capacity of ALL cells histology (Figures 1 and ?and2)2) one-sided screening was chosen, since death rates at a time point in the control Clemizole hydrochloride groups were known and we aimed to detect a difference in one direction. panels are representative of at least 2 impartial experiments. Densitometry was performed using ImageJ.30 The association between gene expression and CNS status was examined by unconditional logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Open in a separate window Physique 1. ZAP70 expression is usually associated with CNS infiltration is usually significant). In the 697-shZAP70 group, 1 cage made up of 3 mice was accidentally eliminated from your experiment on day +2. These animals were not included in the statistical analysis. D: Quantity of leukemic blasts recovered from your CNS of mice xenografted with either JURKAT-shGFP (n=4) or JURKAT-shZAP70 (n=3) (unpaired is usually significant). *26 days, JURKAT-shZAP70 cells (Physique 1C), however, the total quantity of blasts recovered from your CNS of xenografts in controls was significantly higher than those from JURKAT-shZAP70 (Physique 1D). For REH cells, ZAP70 knockdown significantly prolonged the median xenograft survival by 25 days in the REH-shZAP70 group as compared to REH-shGFP (104 79 days, Physique 1E). These data suggest that despite a similar leukemic burden in the periphery, knockdown of ZAP70 hampers the infiltration of 697 and REH BCP-ALL cells into the CNS. In the REH NOD/SCID model, CNS leukemia outweighs peripheral leukemia and mice show low levels of blood, BM and splenic engraftment, but massive CNS infiltration. Our data show that knockdown of Clemizole hydrochloride ZAP70 in REH cells delays CNS infiltration as a cause of xenograft death CCR7,.