No abstract

Cell cycle arrest by FoxO transcription factors involves transcriptional repression of cyclin D, although the exact mechanism remains unclear. In this study, we used the BCR-ABL-expressing cell line BV173 as a model system to investigate the mechanisms whereby FoxO3a regulates cyclin D2 expression. Inhibition of BCR-ABL by STI571 results in down-regulation of cyclin D2 expression, activation of FoxO3a activity, and up-regulation of BCL6 expression. Using reporter gene assays, we demonstrate that STI571, FoxO3a, and BCL6 can repress cyclin D2 transcription through a STAT5/BCL6 site located within the cyclin D2 promoter. We propose that BCR-ABL inhibition leads to FoxO3a activation, which in turn induces the expression of BCL6, culminating in the repression of cyclin D2 transcription through this STAT5/BCL6 site. This process was verified by mobility shift and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to accumulation of BCL6 and down-regulation of cyclin D2 at protein and mRNA levels. Furthermore, silencing of FoxO3a and BCL6 in BCR-ABL-expressing cells abolishes STI571-mediated effects on cyclin D2. This report establishes the signaling events whereby BCR-ABL signals are relayed to cyclin D2 to mediate cell cycle progression and defines a potential mechanism by which FoxO proteins regulate cyclin D2 expression.In mammalian cells, the commitment to divide is made in the G 1 phase of the cell cycle in response to various stimuli, including growth factors. After passing the restriction point at mid-to late G 1 , cells become refractory to growth inhibition signals or do not require growth factors to progress into S phase (37). Progression of eukaryotic cells through the cell cycle is controlled by the two families of G 1 cyclins: (i) D-type cyclins (cyclins D1, D2, and D3) and cyclin E (cyclins E1 and E2) (29, 44) and (ii) the cyclin-dependent kinases (cdk's), their catalytic counterparts. The primary targets of the G 1 cyclin-cdk complexes are the retinoblastoma protein (pRb) family of pocket proteins, consisting of pRb, p107, and p130 (20,27,34). The phosphorylation state of pRb regulates the activity of the E2F family of transcription factors; in their hypophosphorylated forms, the pRb-related pocket proteins associate with members of the E2F family, negatively regulating transcription activity of E2F-regulated genes that are required for entry into the S phase of the cell cycle (15,35,40).In mammals, the phosphatidylinositol 3-kinase/protein kinase B (PI3-K/PKB) pathway is stimulated by a variety of growth factors and cytokines and by cell-matrix interactions, and it controls many biological functions, including cell proliferation, cell survival, and insulin responses (30). Importantly, constitutive activation of the PI3-K pathway facilitates tumor formation by two different mechanisms: it supports S-phase entry, and it confers resistance to apoptotic signals which normally restrict uncontrolled cell growth (49). Recently, it has been demonstrated that the member...

Survival signals elicited by cytokines include the activation of phosphatidylinositol 3-kinase (PI3K), which in turn promotes the activation of protein kinase B (PKB). Recently, PKB has been demonstrated to phosphorylate and inactivate forkhead transcription factor FKHR-L1, a potent inducer of apoptosis. To explore the mechanisms underlying the induction of apoptosis after cytokine withdrawal or FKHR-L1 activation, we used a cell line in which FKHR-L1 activity could be specifically induced. Both cytokine withdrawal and FKHR-L1 activation induced apoptosis, which was preceded by an upregulation in p27KIP1 and a concomitant decrease in cells entering the cell cycle. Induction of apoptosis by both cytokine withdrawal and activation of FKHR-L1 correlated with the disruption of mitochondrial membrane integrity and cytochrome c release. This was preceded by upregulation of the pro-apoptotic Bcl-2 family member Bim. Ectopic expression of an inhibitory mutant of FKHR-L1 substantially reduced the levels of apoptosis observed after cytokine withdrawal. Activation of PKB alone was sufficient to promote cell survival, as measured by maintenance of mitochondrial integrity and the resultant inhibition of effector caspases. Furthermore, hematopoietic stem cells isolated from Bim−/− mice exhibited reduced levels of apoptosis upon inhibition of PI3K/PKB signaling.These data demonstrate that activation of FKHR-L1 alone can recapitulate all known elements of the apoptotic program normally induced by cytokine withdrawal. Thus PI3K/PKB–mediated inhibition of this transcription factor likely provides an important mechanism by which survival factors act to prevent programmed cell death.

In the present study, we aimed to elucidate the mechanism responsible for constitutive NF-jB DNA-binding activity in AML cells. Intervening in aberrant signaling pathway provides a rational approach for in vivo targeting of AML cells. Constitutive NF-jB DNA-binding activity was observed in 16 of 22 (73%) investigated AML cases and was, in general, associated with resistance to spontaneous apoptosis. Indeed, inhibition of NFjB activity by the NF-jB inhibitor SN-50 peptide resulted in enhanced chemotherapy-induced apoptosis. In the majority of cases, constitutive NF-jB activity was mediated by a Ras/PI3 kinase (PI3-K)/protein kinase B (PKB)-mediated pathway. The PI3-K inhibitor Ly294002 and the Ras inhibitor L-744832 both inhibited PKB phosphorylation and NF-jB DNA-binding activity. The constitutive activation of Ras GTP-ase was caused by mutations in the gene encoding for N-Ras in 29% of the cases. The constitutive NF-jB activity could so far not be ascribed to the autocrine production of growth factors or to mutations in the Flt3 receptor, since anti-GM-CSF, -IL-1, -IL6, -TNFa or the tyrosine kinase inhibitor AG1296 did not affect the NF-jB DNAbinding activity. The present study demonstrates that Ras activation is an important pathway for triggering the NF-jB pathway in AML cells.

1 In the present study we investigated a possible role for the p38 mitogen-activated protein (MAP) kinase pathway in mediating nuclear factor-kappa B (NF-kB) transcriptional activity in the erythroleukaemic cell line TF-1. 2 TF-1 cells stimulated with the phosphatase inhibitor okadaic acid (OA) demonstrated enhanced NF-kB and GAL4p65-regulated transcriptional activity which was associated with elevated p38 phosphorylation. However, pretreatment with the p38 MAPK speci®c inhibitor SB203580 (1 mM) or overexpression of kinase-de®cient mutants of MKK3 or MKK6 did not aect OA-enhanced NF-kB transcriptional potency, as determined in transient transfection assays. In fact, 5 and 10 mM SB203580 enhanced rather than inhibited NF-kB-mediated promoter activity by 2 fold, which was independent of phosphorylation of the p65 subunit. 3 The SB203580-mediated increase in NF-kB transcriptional activity was associated with enhanced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK), but not p38 kinase. 4 Overexpression of kinase-de®cient mutants belonging to the ERK1/2, JNK, and p38 pathways showed that only dominant-negative Raf-1 abrogated SB203580-enhanced NF-kB activity. This would implicate the involvement of the ERK1/2 pathway in the enhancing eects of SB203580 on NF-kB-mediated gene transcription. 5 This study demonstrates that the p38 MAP kinase pathway is not involved in the OA-induced activation of NF-kB. SB203580 at higher concentrations activates the ERK pathway, which subsequently enhances NF-kB transcriptional activity.