In Vitro Growth Regulation and the Identification of Potential Signaling Pathways in T (4; 11), Pediatric Pre-B Acute Lymphoblastic Leukemia (ALL).

Abstract

Among the various chromosomal translocations found in pediatric pre-B cell acute leukemias, the t (4; 11) group contains patients of all ages as well as a clustering of children at birth or in the first year of life. As with all 11q23 abnormalities, t (4; 11) fusion confers an extremely poor prognosis. Using cell lines, many recent studies have described important molecular and cellular growth properties of these cells. We have investigated in vitro growth regulation of freshly isolated infant pre-B ALL cells with t (4; 11).Leukemic blasts were affinity purified with anti CD19 antibodies and cultured in the presence of various cytokines including IL-3, IL-1, FLT-3 ligand (FL), SCF, IL-6, IL-7, GM-CSF, PDGF, SDF-1, VEGF, EPO and GCSF. Cell proliferation after four days in culture was quantified by Alamar blue assay. At pg/ ml concentrations, significant growth stimulation was seen in the presence of FL and IL-7. The FLT-3 effect was much less prominent against leukemic cells without t (4; 11). Then we aimed to identify the possible signaling pathways utilized by the FLT-3 mediated stimulation of these cells. Purified blasts were cultured in serum free medium for 24 hours and stimulated for 0, 5 and 10 minutes with 10 pg/ml of FL. These cells were subsequently lysed and total cellular extracts were analyzed by an antibody array technique. The arrays used consisted of antibodies against a selection of fifty proteins involved in all known signaling pathways. Changes in the amount of phosphorylated proteins following FL treatment were identified by enzyme conjugated anti phospho specific antibodies. A number of reproducible FL specific changes were observed. These include increased Ets-1/2, JNK1, 2,3, p38MAPK and Src. We describe the detail analysis of the signaling pathways potentially utilized by these cells and illustrate the use of this experimental approach to identify possible targeted therapeutic agents against this particular form of leukemia.