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)

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.

Emotional stress affects cellular integrity in many tissues including the heart. Much less is known about the effects of social stress. We studied the effect of emotional (immobilization with or without cold exposure) or social (intermale confrontation) stress in mice. Tissue injury was measured by means of the release of enzyme activities to blood plasma: lactate dehydrogenase (LDH), creatine kinase (CK), aspartate transaminase (AST), and alanine transaminase (ALT). Tape-immobilization increased all these activities in the plasma. AST-ALT ratio was also increased in these animals. Electrophoretic analysis of CK isoenzymes showed the appearance of CK-MB. These results indicate that the heart was injured in immobilized mice. Analysis of LDH isoenzymes and measurement of alpha-hydroxybutyrate dehydrogenase (HBDH) activity suggests that other tissues, in addition to the heart, contribute to the increase in plasma LDH activity. Restraint in small cylinders increased plasma LDH, CK, AST, and ALT activities, but to lower levels than in tape immobilization. Because the decrease in liver glycogen and the increase in plasma epidermal growth factor (EGF) were also smaller in restraint than in the tape-immobilization model of emotional stress, we conclude that the former is a less intense stressor than the latter. Cold exposure during the restraint period altered the early responses to stress (it enhanced liver glycogen decrease, but abolished the increase in plasma EGF concentration). Cold exposure during restraint enhanced heart injury, as revealed by the greater increase in CK and AST activities. Intermale confrontation progressively decreased liver glycogen content. Plasma EGF concentration increased (to near 100 nM from a resting value of 0.1 nM) until 60 minutes, and decreased thereafter. Confrontation also affected cellular integrity in some tissues, as indicated by the rise in plasma LDH activity. However, in this type of stress, the heart appeared to be specifically protected because there was no increase in plasma CK activity, and both AST and ALT increased, but the AST-ALT ratio remained constant. Habituation to restraint (1 h/d, 4 days) made mice resistant to restraint-induced tissue injury as indicated by the lack of an increase in plasma LDH, CK, AST, or ALT activities. Similar general protection against homotypic stress-induced injury was observed in mice habituated to intermale confrontation.

No abstract

By imposing a replicative defect in most somatic cells, gradual telomere attrition during aging is thought to progressively impair cellular function and viability and may contribute to age-related disease. Immune cells play important roles in all phases of atherosclerosis, a multifactorial disease that prevails within the elderly. Because shorter telomeres have been found in circulating blood leukocytes of human patients with advanced coronary atherosclerosis, it has been suggested that telomere shortening may predispose the organism to atheroma development. In this study, we assessed the impact of telomere attrition on atherogenesis induced by dietary cholesterol in apolipoprotein E (apoE)-deficient mice, a well-established model of experimental atherosclerosis that recapitulates important aspects of the human disease. Our study shows that late-generation mice doubly deficient in apoE and telomerase RNA experience telomere attrition and a substantial reduction of atherosclerosis compared with control mice with intact telomerase, in spite of sustained hypercholesterolemia in response to the atherogenic diet. Short telomeres impaired the proliferation of both lymphocytes and macrophages, an important step in atherosclerosis development. Therefore, telomere exhaustion resulting in replicative immunosenescence may serve as a mechanism for restricting atheroma progression.