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Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification by Philipp le Coutre, Elena Tassi, Marileila Varella-Garcia, Rossella Barni, Luca Mologni, Gonçalo Cabrita, Edoardo Marchesi, Rosanna Supino, and Carlo Gambacorti-Passerini Blood Volume 95(5):1758-1766 March 1, 2000 ©2000 by American Society of Hematology Proliferative activity as determined by 3HTdR uptake assay.104 cells from LAMA84 and LAMA84R were incubated in 6 replicates in the presence of various concentrations of STI571 ranging from 0.1 to 10 μM. Philipp le Coutre et al. Blood 2000;95:1758-1766 ©2000 by American Society of Hematology Induction of apoptosis in LAMA84 and LAMA84R cells.Cells (0.5 × 106) were incubated with 1 μM STI571 and cultured over 28 hours. Philipp le Coutre et al. Blood 2000;95:1758-1766 ©2000 by American Society of Hematology Induction of caspase 3–like activity in LAMA84 and LAMA84R.The cleavage of the caspase 3 substrate DEVD-amc was evaluated in a fluorimetric assay, as described in “Materials and Methods.” Caspase 3–like activity was detected by reading samples spectrophotof... Philipp le Coutre et al. Blood 2000;95:1758-1766 ©2000 by American Society of Hematology Philipp le Coutre et al. Blood 2000;95:1758-1766 ©2000 by American Society of Hematology Philipp le Coutre et al. Blood 2000;95:1758-1766 ©2000 by American Society of Hematology Philipp le Coutre et al. Blood 2000;95:1758-1766 ©2000 by American Society of Hematology Sequence alignment of the bcr/abl ATP-binding domain.The predicted nucleotidic sequence of the ATP-binding domain ofbcr/abl (Gene Bank accession number: M14752) was aligned with the corresponding sequences obtained from the LAMA84 and LAMA84Rbcr/abl cDNAs. Philipp le Coutre et al. Blood 2000;95:1758-1766 ©2000 by American Society of Hematology Determination of BCR/ABL mRNA in LAMA84 and LAMA84R cells.Samples containing 25 μg of total RNA per lane, extracted from LAMA84 and LAMA84R, were fractionated and analyzed by Northern blotting. Philipp le Coutre et al. Blood 2000;95:1758-1766 ©2000 by American Society of Hematology FISH analysis of LAMA84 and LAMA84R cell lines.(A) Interphase nuclei from LAMA84 displaying 2, 3, and 5 copies of the fused BCR/ABL signal indicating the BCR/ABL chimeric gene. Philipp le Coutre et al. Blood 2000;95:1758-1766 ©2000 by American Society of Hematology Sensitivity of LAMA84 and LAMA84R to doxorubicin.The graph shows the sensitivity of LAMA84 (diamonds) and LAMA84R (squares) to doxorubicin, calculated by cell count. Philipp le Coutre et al. Blood 2000;95:1758-1766 ©2000 by American Society of Hematology