Downregulation of beta1,4-galactosyltransferase 1 inhibits CDK11(p58)-mediated apoptosis induced by cycloheximide.

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34(cdc2)-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. The mechanism that CDK11(p58) induces apoptosis is not clear. Some evidences suggested beta1,4-galactosyltransferase 1 (beta1,4-GT 1) might participate in apoptosis induced by CDK11(p58). In this study, we demonstrated that ectopically expressed beta1,4-GT 1 increased CDK11(p58)-mediated apoptosis induced by cycloheximide (CHX). In contrast, RNAi-mediated knockdown of beta1,4-GT 1 effectively inhibited apoptosis induced by CHX in CDK11(p58)-overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-G1 phase was decreased. Knock down of beta1,4-GT 1 also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore, the cleavage of CDK11(p58) by caspase-3 was reduced. We proposed that beta1,4-GT 1 might contribute to the pro-apoptotic effect of CDK11(p58). This may represent a new mechanism of beta1,4-GT 1 in CHX-induced apoptosis of CDK11(p58)-overexpressing cells.

The C-terminal kinase domain of the p34cdc2-related PITSLRE protein kinase (p110C) associates with p21-activated kinase 1 and inhibits its activity during anoikis.

The PITSLRE protein kinases are parts of the large family of p34cdc2-related kinases. During apoptosis induced by some stimuli, specific PITSLRE isoforms are cleaved by caspase to produce a protein that contains the C-terminal kinase domain of the PITSLRE proteins (p110C). The p110C induces apoptosis when it is ectopically expressed in Chinese hamster ovary cells. In our study, similar induction of this p110C was observed during anoikis in NIH3T3 cells. To investigate the molecular mechanism of apoptosis mediated by p110C, we used the yeast two-hybrid system to screen a human fetal liver cDNA library and identified p21-activated kinase 1 (PAK1) as an interacting partner of p110C. The association of p110C with PAK1 was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscope analysis. The interaction of p110C with PAK1 occurred within the residues 210-332 of PAK1. Neither association between p58PITSLRE or p110PITSLRE and PAK1 nor association between p110C and PAK2 or PAK3 was observed. Anoikis was increased and PAK1 activity was inhibited when NIH3T3 cells were transfected with p110C. Furthermore, the binding of p110C with PAK1 and inhibition of PAK1 activity were also observed during anoikis. Taken together, these data suggested that PAK1 might participate in the apoptotic pathway mediated by p110C.

Effects of the aurora kinase inhibitors AZD1152-HQPA and ZM447439 on growth arrest and polyploidy in acute myeloid leukemia cell lines and primary blasts.

BACKGROUND:
Aurora kinases play an essential role in the orchestration of chromosome separation and cytokinesis during mitosis. Small-molecule inhibition of the aurora kinases has been shown to result in inhibition of cell division, phosphorylation of histone H3 and the induction of apoptosis in a number of cell systems. These characteristics have led aurora kinase inhibitors to be considered as potential therapeutic agents.
DESIGN AND METHODS:
Aurora kinase gene expression profiles were assessed in 101 samples from patients with acute myeloid leukemia. Subsequently, aurora kinase inhibitors were investigated for their in vitro effects on cell viability, histone H3 phosphorylation, cell cycle and morphology in acute myeloid leukemia cell lines and primary acute myeloid leukemia samples.
RESULTS:
The aurora kinase inhibitors AZD1152-HQPA and ZM447439 induced growth arrest and the accumulation of hyperploid cells in acute myeloid leukemia cell lines and primary acute myeloid leukemia cultures. Furthermore, both agents inhibited histone H3 phosphorylation and this preceded perturbations in cell cycle and the induction of apoptosis. Single cell cloning assays were performed on diploid and polyploid cells to investigate their colony-forming capacities. Although the polyploid cells showed a reduced capacity for colony formation when compared with their diploid counterparts, they were consistently able to form colonies.
CONCLUSIONS:
AZD1152-HQPA- and ZM447439 are effective apoptosis-inducing agents in acute myeloid leukemia cell lines and primary acute myeloid leukemia cultures. However, their propensity to induce polyploidy does not inevitably result in apoptosis.

The frequency of JAK2 exon 12 mutations in idiopathic erythrocytosis patients with low serum erythropoietin levels

Idiopathic erythrocytosis (IE) is characterized by erythrocytosis in the absence of megakaryocytic or granulocytic hyperplasia, and is associated with variable serum erythropoietin (Epo) levels. Most patients with IE lack the JAK2 V617F mutation that occurs in the majority of polycythemia vera patients. Four novel JAK2 mutant alleles have recently been described in patients with V617F-negative myeloproliferative disorders presenting with erythrocytosis. The aims of this study were to assess the prevalence of JAK2 exon 12 mutations in IE patients, and to determine the associated clinicopathological features.

Src and ADAM-17-Mediated Shedding of Transforming Growth Factor-alpha Is a Mechanism of Acute Resistance to TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) has emerged as a promising anticancer agent. However, resistance to TRAIL is likely to be a major problem, and sensitization of cancer cells to TRAIL may therefore be an important anticancer strategy. In this study, we examined the effect of the epidermal growth factor receptor (EGFR)tyrosine kinase inhibitor (TKI) gefitinib and a human epidermal receptor 2 (HER2)-TKI (M578440) on the sensitivity of human colorectal cancer (CRC) cell lines to recombinant human TRAIL (rhTRAIL). A synergistic interaction between rhTRAIL and gefitinib and rhTRAIL and M578440 was observed in both rhTRAIL-sensitive and resistant CRC cells. This synergy correlated with an increase in EGFR and HER2 activation after rhTRAIL treatment. Furthermore, treatment of CRC cells with rhTRAIL resulted in activation of the Src family kinases (SFK). Importantly, we found that rhTRAIL treatment induced shedding of transforming growth factor-alpha (TGF-alpha) that was dependent on SFK activity and the protease ADAM-17. Moreover, this shedding of TGF-alpha was critical for rhTRAIL-induced activation of EGFR. In support of this, SFK inhibitors and small interfering RNAs targeting ADAM-17 and TGF-alpha also sensitized CRC cells to rhTRAIL-mediated apoptosis. Taken together, our findings indicate that both rhTRAIL-sensitive and resistant CRC cells respond to rhTRAIL treatment by activating an EGFR/HER2-mediated survival response and that these cells can be sensitized to rhTRAIL using EGFR/HER2-targeted therapies. Furthermore, this acute response to rhTRAIL is regulated by SFK-mediated and ADAM-17-mediated shedding of TGF-alpha, such that targeting SFKs or inhibiting ADAM-17, in combination with rhTRAIL, may enhance the response of CRC tumors to rhTRAIL. [Cancer Res 2008;68(20):8312-21]

Regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) by src involves tyrosine phosphorylation of PDK1 and Src homology 2 domain binding

3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr(9) and Tyr(373/376)) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1- Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti- phospho-Tyr9 antibodies showed that the level of Tyr9 phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr9, distinct from Tyr373/376, is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr9 phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.

Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane

The PKB (protein kinase B, also called Akt) family of protein kinases plays a key role in insulin signaling, cellular survival, and transformation. PKB is activated by phosphorylation on residues threonine 308, by the protein kinase PDK1, and Serine 473, by a putative serine 473 kinase. Several protein binding partners for PKB have been identified. Here, we describe a protein partner for PKB alpha termed CTMP, or carboxyl-terminal modulator protein, that binds specifically to the carboxyl-terminal regulatory domain of PKB alpha at the plasma membrane. Binding of CTMP reduces the activity of PKB alpha by inhibiting phosphorylation on serine 473 and threonine 308. Moreover, CTMP expression reverts the phenotype of v-Akt-transformed cells examined under a number of criteria including cell morphology, growth rate, and in vivo tumorigenesis. These findings identify CTMP as a negative regulatory component of the pathway controlling PKB activity.

USP17 Regulates Ras Activation and Cell Proliferation by Blocking RCE1 Activity

The proto-oncogene Ras undergoes a series of post-translational modifications at its carboxyl-terminal CAAX motif that are essential for its proper membrane localization and function. One step in this process is the cleavage of the CAAX motif by the enzyme Ras-converting enzyme 1 (RCE1). Here we show that the deubiquitinating enzyme USP17 negatively regulates the activity of RCE1. We demonstrate that USP17 expression blocks Ras membrane localization and activation, thereby inhibiting phosphorylation of the downstream kinases MEK and ERK. Furthermore, we show that this effect is caused by the loss of RCE1 catalytic activity as a result of its deubiquitination by USP17. We also show that USP17 and RCE1 co-localize at the endoplasmic reticulum and that USP17 cannot block proliferation or Ras membrane localization in RCE1 null cells. These studies demonstrate that USP17 modulates Ras processing and activation, at least in part, by regulating RCE1 activity.

Beta2 integrins target Rap GTPases to the plasma membrane by means of degranulation.

We report the novel observation that engagement of ß2 integrins on human neutrophils is accompanied by increased levels of the small GTPases Rap1 and Rap2 in a membrane-enriched fraction and a concomitant decrease of these proteins in a granule-enriched fraction. In parallel, we observed a similar time-dependent decrease of gelatinase B (a marker of specific and gelatinase B-containing granules) but not myeloperoxidase (a marker of azurophil granules) in the granule fraction, and release of lactoferrin (a marker of specific granules) in the extracellular medium. Furthermore, inhibition of Src tyrosine kinases, or phosphoinositide 3-kinase with PP1 or LY294002, respectively, blocked ß2 integrin-induced degranulation and the redistribution of Rap1 and Rap2 to a membrane-enriched fraction. Consequently, the ß2 integrin-dependent exocytosis of specific and gelatinase B-containing granules occurs via a Src tyrosine kinase/phosphoinositide 3-kinase signaling pathway and is responsible for the translocation of Rap1 and Rap2 to the plasma membrane in human neutrophils.

3-anilino-4-arylmaleimides: Potent and selective inhibitors of glycogen synthase kinase-3 (GSK-3)

Potent 3-anilino-4-arylmaleimide glycogen synthase kinase-3 (GSK-3) inhibitors have been prepared using automated array methodology. A number of these are highly selective, having little inhibitory potency against more than 20 other protein kinases. (C) 2001 Elsevier Science Ltd. All rights reserved.

Erythropoietin-Induced Activation of the JAK2/STAT5, PI3K/Akt, and Ras/ERK Pathways Promotes Malignant Cell Behavior in a Modified Breast Cancer Cell Line

Abstract Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA-mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread.

SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr(346/356/366) phosphorylation

Studies of HeLa cells and serum- and glucocorticoid-regulated kinase 1 (SGK1) knockout mice identified threonine residues in the n-myc downstream-regulated gene 1 protein (NDRG1-Thr(346/356/366)) that are phosphorylated by SGK1 but not by related kinases (Murray et al., Biochem J 385:1-12, 2005). We have, therefore, monitored the phosphorylation of NDRG1-Thr(346/356/366) in order to explore the changes in SGK1 activity associated with the induction and regulation of the glucocorticoid-dependent Na+ conductance (G (Na)) in human airway epithelial cells. Transient expression of active (SGK1-S422D) and inactive (SGK1-K127A) SGK1 mutants confirmed that activating SGK1 stimulates NDRG1-Thr(346/356/366) phosphorylation. Although G (Na) is negligible in hormone-deprived cells, these cells displayed basal SGK1 activity that was sensitive to LY294002, an inhibitor of 3-phosphatidylinositol phosphate kinase (PI3K). Dexamethasone (0.2 mu M) acutely activated SGK1 and the peak of this response (2-3 h) coincided with the induction of G (Na), and both responses were PI3K-dependent. While these data suggest that SGK1 might mediate the rise in G (Na), transient expression of the inactive SGK1-K127A mutant did not affect the hormonal induction of G (Na) but did suppress the activation of SGK1. Dexamethasone-treated cells grown on permeable supports formed confluent epithelial sheets that generated short circuit current due to electrogenic Na+ absorption. Forskolin and insulin both stimulated this current and the response to insulin, but not forskolin, was LY294002-sensitive and associated with the activation of SGK1. While these data suggest that SGK1 is involved in the control of G (Na), its role may be minor, which could explain why sgk1 knockout has different effects upon different tissues.

The Ectocarpus genome and the independent evolution of multicellularity in brown algae

Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related(1). These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae(2-5), closely related to the kelps(6,7) (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic(2) approaches to explore these and other(4,5) aspects of brown algal biology further.

Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha

Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane(1). Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses(1). The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class I-A Pi3ks (ref. 2). Mice lacking only the p85a isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants(3). Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites, We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class I-A Pi3k catalytic subunits: nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.

Two routes to leukemic transformation after a JAK2 mutation-positive myeloproliferative neoplasm

Acute myeloid leukemia (AML) may follow a JAK2-positive myeloproliferative neoplasm (MPN), although the mechanisms of disease evolution, often involving loss of mutant JAK2, remain obscure. We studied 16 patients with JAK2-mutant (7 of 16) or JAK2 wild-type (9 of 16) AML after a JAK2-mutant MPN. Primary myelofibrosis or myelofibrotic transformation preceded all 7 JAK2-mutant but only 1 of 9 JAK2 wild-type AMLs (P = .001), implying that JAK2-mutant AML is preceded by mutation(s) that give rise to a "myelofibrosis" phenotype. Loss of the JAK2 mutation by mitotic recombination, gene conversion, or deletion was excluded in all wild-type AMLs. A search for additional mutations identified alterations of RUNX1, WT1, TP53, CBL, NRAS, and TET2, without significant differences between JAK2-mutant and wild-type leukemias. In 4 patients, mutations in TP53, CBL, or TET2 were present in JAK2 wild-type leukemic blasts but absent from the JAK2-mutant MPN. By contrast in a chronic-phase patient, clones harboring mutations in JAK2 or MPL represented the progeny of a shared TET2-mutant ancestral clone. These results indicate that different pathogenetic mechanisms underlie transformation to JAK2 wild-type and JAK2-mutant AML, show that TET2 mutations may be present in a clone distinct from that harboring a JAK2 mutation, and emphasize the clonal heterogeneity of the MPNs.