Bortezomib decreases Rb phosphorylation and induces caspase-dependent apoptosis in Imatinib-sensitive and -resistant Bcr-Abl1-expressing cells

The use of c-abl-specific inhibitors such as Imatinib (IM) or Dasatinib has revolutionized the treatment of chronic myeloid leukemia (CML). However, a significant percentage of patients become resistant to IM. In this report, we have analyzed the possibility of using the proteasome as a molecular target in CML. Our results show that cells that express Bcr-Abl1 are more sensitive to the inhibition of the proteasome with Bortezomib (Btz) than control cells. This treatment reduces the proliferation of Bcr-Abl1- expressing cells, by inactivating NF-jB2 and decreasing the phosphorylation of Rb, eventually leading to an increase in caspase-dependent apoptosis. Furthermore, we show that Btz also induces cell-cycle arrest and apoptosis in cells expressing Bcr-Abl1 mutants that are resistant to IM. These results unravel a new molecular target of Btz, that is the Rb pathway, and open new possibilities in the treatment of CML especially for patients that become resistant to IM because of the presence of the T315I mutation

BCR-ABL induces the expression of Skp2 through the PI3K pathway to promote p27Kip1 degradation and proliferation of chronic myelogenous leukemia cells

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 p27(Kip1) 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 p27(Kip1) at the level of transcription. We further show that BCR-ABL also regulates p27(Kip1) protein levels by increasing its degradation by the proteasome. This degradation depends on the ubiquitinylation of p27(Kip1) by Skp2-containing SFC complexes: silencing the expression of Skp2 with a small interfering RNA results in the accumulation of p27(Kip1). We also demonstrate that BCR-ABL cells show transcriptional up-regulation of Skp2. Finally, expression of a p27(Kip1) mutant unable of being recognized by Skp2 results in inhibition of proliferation of BCR-ABL cells, indicating that the degradation of p27(Kip1) 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 p27(Kip1) and provide a rationale for the use of inhibitors of the proteasome in patients with BCR-ABL leukemias

RhoE Deficiency Produces Postnatal Lethality, Profound Motor Deficits and Neurodevelopmental Delay in Mice

Rnd proteins are a subfamily of Rho GTPases involved in the control of actin cytoskeleton dynamics and other cell functions such as motility, proliferation and survival. Unlike other members of the Rho family, Rnd proteins lack GTPase activity and therefore remain constitutively active. We have recently described that RhoE/Rnd3 is expressed in the Central Nervous System and that it has a role in promoting neurite formation. Despite their possible relevance during development, the role of Rnd proteins in vivo is not known. To get insight into the in vivo function of RhoE we have generated mice lacking RhoE expression by an exon trapping cassette. RhoE null mice (RhoE gt/gt) are smaller at birth, display growth retardation and early postnatal death since only half of RhoE gt/gt mice survive beyond postnatal day (PD) 15 and 100% are dead by PD 29. RhoE gt/gt mice show an abnormal body position with profound motor impairment and impaired performance in most neurobehavioral tests. Null mutant mice are hypoactive, show an immature locomotor pattern and display a significant delay in the appearance of the hindlimb mature responses. Moreover, they perform worse than the control littermates in the wire suspension, vertical climbing and clinging, righting reflex and negative geotaxis tests. Also, RhoE ablation results in a delay of neuromuscular maturation and in a reduction in the number of spinal motor neurons. Finally, RhoE gt/gt mice lack the common peroneal nerve and, consequently, show a complete atrophy of the target muscles. This is the first model to study the in vivo functions of a member of the Rnd subfamily of proteins, revealing the important role of Rnd3/RhoE in the normal development and suggesting the possible involvement of this protein in neurological disorders

Bortezomib decreases Rb phosphorylation and induces caspase-dependent apoptosis in Imatinib-sensitive and -resistant Bcr-Abl1-expressing cells

The use of c-abl-specific inhibitors such as Imatinib (IM) or Dasatinib has revolutionized the treatment of chronic myeloid leukemia (CML). However, a significant percentage of patients become resistant to IM. In this report, we have analyzed the possibility of using the proteasome as a molecular target in CML. Our results show that cells that express Bcr-Abl1 are more sensitive to the inhibition of the proteasome with Bortezomib (Btz) than control cells. This treatment reduces the proliferation of Bcr-Abl1- expressing cells, by inactivating NF-jB2 and decreasing the phosphorylation of Rb, eventually leading to an increase in caspase-dependent apoptosis. Furthermore, we show that Btz also induces cell-cycle arrest and apoptosis in cells expressing Bcr-Abl1 mutants that are resistant to IM. These results unravel a new molecular target of Btz, that is the Rb pathway, and open new possibilities in the treatment of CML especially for patients that become resistant to IM because of the presence of the T315I mutation

BCR-ABL induces the expression of Skp2 through the PI3K pathway to promote p27Kip1 degradation and proliferation of chronic myelogenous leukemia cells

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 p27(Kip1) 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 p27(Kip1) at the level of transcription. We further show that BCR-ABL also regulates p27(Kip1) protein levels by increasing its degradation by the proteasome. This degradation depends on the ubiquitinylation of p27(Kip1) by Skp2-containing SFC complexes: silencing the expression of Skp2 with a small interfering RNA results in the accumulation of p27(Kip1). We also demonstrate that BCR-ABL cells show transcriptional up-regulation of Skp2. Finally, expression of a p27(Kip1) mutant unable of being recognized by Skp2 results in inhibition of proliferation of BCR-ABL cells, indicating that the degradation of p27(Kip1) 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 p27(Kip1) and provide a rationale for the use of inhibitors of the proteasome in patients with BCR-ABL leukemias