Activation of PI 3-kinase in malignant lymphoblasts from mice with p190-induced B-lymphoid leukemia.

(A) Whitlock-Witte (W/W) cultures. Primary bone marrow cells were transduced with BCR/ABL wild-type and SH2 FLVRES (RK = R552K for p190 and R1053K for p210) and deletion (ΔSH2) mutants and cultured in vitro as described in “Materials and methods.” Stromal-independent populations of lymphoblasts were readily established, cell extracts prepared 12 to 14 days after transduction, and assayed for PI 3-kinase activity in antiphosphotyrosine immunoprecipitates. As controls, Ba/F3 parental cells or a population transformed with p210 BCR/ABL(Ba/F3-210), and B220⁺ splenocytes isolated from Balb/c mice 8 weeks after transplantation with untransduced donor marrow (B220⁺ spl.) were used. Results are representative of 3 independent experiments and expressed as fold increase in PI 3-kinase activity over that present in parental Ba/F3 cells. (B) Primary malignant lymphoblasts from diseased mice. Lysates from cells isolated from pleural effusion (left) or malignant lymph nodes (right) were assayed for PI 3-kinase activity in antiphosphotyrosine immunoprecipitates. Wright-Giemsa staining of cytospin preparations indicated that all populations were more than 80% lymphoblasts (data not shown). Results represent the mean and standard error of PI 3-kinase activity in tumor cell populations from several independent animals for each p190 genotype, as indicated by the numbers at the bottom, expressed as fold increase over control B220⁺splenocytes (B220⁺ spl.). The difference in mean lymph node PI 3-kinase activity between p190 wild-type and p190 ΔSH2 was not significant (P = .66, Student t test). (C) Whole cell lysate assay. PI 3-kinase activity was measured in diluted whole cell lysates from lymph node-derived lymphoblasts and control B220⁺ splenocytes, as described in “Materials and methods.” Results represent the mean and standard error for PI 3-kinase activity in lymphoblasts from several independent mice, as indicated, expressed as fold increase over B220⁺splenocytes.