3 research outputs found
The leukemogenic potential of t(9;22) fusion proteins.
<p>(A) Schematic representation of the experimental procedure. Sca1<sup>+</sup> bone marrow cells were infected with the indicated retroviruses and inoculated into sub-lethally irradiated mice. Empty vector-transduced cells were used as control; (B) Kaplan Maier curves present the probability of survival upon primary induction of leukemia and re-transplantation of leukemic cells in order to induce secondary (II) leukemia; (C) May-Grünwald-Giemsa staining of cytospins from BM and spleens of p185<sup><i>BCR/ABL</i></sup> and p96+p185-positive leukemia of one representative mouse in each group; (D) Expression of differentiation specific surface markers (Mac1: monocytes- macrophage, Gr1: granulocytes and B220: mature B-cells) of one representative mouse in p185<sup><i>BCR/ABL</i></sup> and <i>p96</i><sup><i>ABL/BCR</i></sup> + p185<sup><i>BCR/ABL</i></sup> (p96+p185) groups. (E) Co-expression of differentiation specific surface markers (Mac1/Gr-1—myeloid leukemia; B220/CD19: B-cell leukemia) of one representative mouse in p185<sup><i>BCR/ABL</i></sup> and <i>p96</i><sup><i>ABL/BCR</i></sup> + p185<sup><i>BCR/ABL</i></sup> (p96+p185) groups and secondary transplanted sup.</p
Targeting p96<sup><i>ABL/BCR</i></sup> in Ph<sup>+</sup> ALL cells.
<p>(A) SupB15 and K562 cells were lentivirally transduced with shRNA against p96<sup><i>ABL/BCR</i></sup> (siR961 and siR962) and a control shRNA (NTC). The expression of p96<sup><i>ABL/BCR</i></sup> and/or BCR was detected by immunoblotting using anti-BCR antibody. Tubulin was used as loading control. Proliferation was measured using XTT-assay after 3 days. One representative experiment in triplicates ± SD of at least three yielding similar results is given; (B) The effect of targeting p96<sup><i>ABL/BCR</i></sup> by shRNA in SupB15 on STAT5 and ERK1/2 pathway was detected using the indicated antibodies; (C) Down-regulation of p96<sup><i>ABL/BCR</i></sup> in Ph<sup>+</sup> ALL PD-LTCs by shRNA. Ph<sup>+</sup> ALL PD-LTCs—PH: fully TKI-responsive; BV: TKI-resistant; as controls were used: HP (Ph<sup>-</sup> ALL patient) and VG: t(12;9)-TEL/ABL-positive ALL. The effect of shRNAs on the expression of p96<sup><i>ABL/BCR</i></sup> was tested by immunoblotting using the indicated antibodies and by q-RT-PCR for PH and BV. The Ct values were normalized to that of GAPDH and results are represented as 2<sup>-ΔΔCt</sup>. Proliferation was measured by XTT-assay. The mean of at least experiments is given ± SD.</p
Stem cell colonogenic potential of t(9;22) fusion proteins.
<p>(A) Experimental strategy for studying the influence of t(9;22) fusion proteins on the biology of murine HSCs. Sca1<sup>+</sup>/lin<sup>-</sup> bone marrow (BM) cells were infected with the indicated retroviruses and maintained for 9 days in liquid culture supplemented with the indicated growth factors. 1 x 10<sup>4</sup> cells were inoculated into lethally irradiated recipients that were sacrificed at day 12 after transplantation; (B) Number of colonies in the spleens (n = 3); the experiment was performed a total of three times with similar results. One representative experiment is given; (C) Gene expression profile induced by t(9;22) fusion proteins in spleen from the CFU-S12. Clustering was done by selecting genes with the highest SD and sorted according to the similarity in expression level; (D) <i>Seven representative cellular pathways known to be influenced by BCR/ABL related to cell cycle regulation</i>, <i>proliferation and apoptosis are presented here</i>. <i>The numbers indicate the number of differentially expressed genes between p185</i><sup><i>BCR/ABL</i></sup><i>and p96</i><sup><i>ABL/BCR</i></sup> + p185<sup><i>BCR/ABL</i></sup> (p96+p185)-positive cells from the total number of the genes (related to each of the pathways); (E) Total RNA was isolated from spleens from the CFU-S12. The expression levels of Tp53, Gadd45α, and Cdkn1a were analyzed using q-RT-PCR. The Ct values were normalized to that of Gapdh and results are represented as 2<sup>-ΔΔCt</sup>. The mean of three independent experiments each done in triplicates is given ± SD; (F) PH and BV were lentivirally transduced with shRNA against p96<sup><i>ABL/BCR</i></sup> (siR961 and siR962) and a control shRNA (NTC). The expression of GADD45α was detected by q-RT-PCR. The Ct values were normalized to that of GAPDH and results are represented as 2<sup>-ΔΔCt</sup>.</p