Bcr-Abl–expressing leukemic cells are highly resistant to apoptosis induced by chemotherapeutic drugs. Although a number of signaling molecules have been shown to be activated by the Bcr-Abl kinase, the antiapoptotic pathway triggered by this oncogene has not been elucidated. Here, we show that the interleukin 3-independent expression of the antiapoptotic protein, Bcl-xL, is induced by Bcr-Abl through activation of signal transducer and activator of transcription (Stat)5. Inhibition of the Bcr-Abl kinase activity in Bcr-Abl–expressing cell lines and CD34+ cells from chronic myelogenous leukemia (CML) patients induces apoptosis by suppressing the capacity of Stat5 to interact with the bcl-x promoter. Interestingly, after inhibition of the Bcr-Abl kinase, the expression of Bcl-xL is downregulated more rapidly in chronic phase than in blast crisis CML cells, suggesting an involvement of this protein in disease progression. Overall, we describe a novel antiapoptotic pathway triggered by Bcr-Abl that may contribute to the resistance of CML cells to undergo apoptosis.
Chronic myelogenous leukemia (CML) is characterized by the expression of the BCR-ABL tyrosine kinase, which results in increased cell proliferation and inhibition of apoptosis. In this study, we show in both BCR-ABL cells (Mo7e-p210 and BaF/3-p210) and primary CML CD34+ cells that STI571 inhibition of BCR-ABL tyrosine kinase activity results in a G 1 cell cycle arrest mediated by the PI3K pathway. This arrest is associated with a nuclear accumulation of p27Kip1 and down-regulation of cyclins D and E. As a result, there is a reduction of the cyclin E/Cdk2 kinase activity and of the retinoblastoma protein phosphorylation. By quantitative reverse transcription-PCR we show that BCR-ABL/PI3K regulates the expression of p27Kip1 at the level of transcription. We further show that BCR-ABL also regulates p27Kip1 protein levels by increasing its degradation by the proteasome. This degradation depends on the ubiquitinylation of p27Kip1 by Skp2-containing SFC complexes: silencing the expression of Skp2 with a small interfering RNA results in the accumulation of p27Kip1 . We also demonstrate that BCR-ABL cells show transcriptional up-regulation of Skp2. Finally, expression of a p27Kip1 mutant unable of being recognized by Skp2 results in inhibition of proliferation of BCR-ABL cells, indicating that the degradation of p27Kip1 contributes to the pathogenesis of CML. In conclusion, these results suggest that BCR-ABL regulates cell cycle in CML cells at least in part by inducing proteasome-mediated degradation of the cell cycle inhibitor p27Kip1 and provide a rationale for the use of inhibitors of the proteasome in patients with BCR-ABL leukemias. (Cancer Res 2005; 65(8): 3264-72)
Summary:Despite the wide use of G-CSF for mobilization of PBPC the best dose and schedule of G-CSF has not been definitively established. In this study we have compared three different schedules of G-CSF for mobilization of PBPC in normal donors including a single daily dose of 10 g/kg/day for 5 days (21 donors) and doses of 6 (21 donors) or 8 g/kg/12 h (6 donors) for 5 days. We demonstrate that G-CSF at doses of 6 and 8 g/kg/12 h mobilizes significantly more CD34 ؉ cells/ml of blood (83.3 ؎ 6.7 and 121 ؎ 6.9, respectively) than 10 g/kg/day (71.6 ؎ 6.5). Mobilization with 6 or 8 g/kg/12 h of G-CSF was also associated with collection of significantly more CD34؉ cells in comparison with 10 g/kg/24 h (2.24 ؎ 1.2 and 2.46 ؎ 1.22 vs 1.15 ؎ 0.8 CD34 ؉ cells/kg of donor/blood volume). PBPC collection was associated with a significant decrease in platelet count which was not significantly different between the three groups. Ten days after the last PBPC collection platelet counts were within normal limits while there was a decrease in WBC and ANC. We conclude that G-CSF administered every 12 h at doses of 6 g/kg provides better CD34 ؉ cell yield than 10 g/kg once a day in normal donors which may translate into a decrease in the number of aphereses required to obtain enough numbers of CD34 ؉ cells for allogeneic PBPC transplant.
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