RÉSUMÉ
Constitutively active casein kinase 2 (CK2) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL). CK2 phosphorylates PTEN (phosphatase and tensin homolog) tumor suppressor, resulting in PTEN stabilization and functional inactivation. Downregulation of PTEN activity has an impact on PI3K/Akt/mTOR signaling, which is of fundamental importance for T-ALL cell survival. These observations lend compelling weight to the application of CK2 inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of CX-4945-a novel, highly specific, orally available, ATP-competitive inhibitor of CK2α. We show that CX-4945 treatment induced apoptosis in T-ALL cell lines and patient T lymphoblasts. CX-4945 downregulated PI3K/Akt/mTOR signaling in leukemic cells. Notably, CX-4945 affected the unfolded protein response (UPR), as demonstrated by a significant decrease in the levels of the main UPR regulator GRP78/BIP, and led to apoptosis via upregulation of the ER stress/UPR cell death mediators IRE1α and CHOP. In vivo administration of CX-4945 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth. Our findings indicate that modulation of the ER stress/UPR signaling through CK2 inhibition could be exploited for inducing apoptosis in T-ALL cells and that CX-4945 may be an efficient treatment for those T-ALLs displaying upregulation of CK2α/PI3K/Akt/mTOR signaling.
Sujet(s)
Antinéoplasiques/usage thérapeutique , Casein Kinase II/antagonistes et inhibiteurs , Naphtyridines/usage thérapeutique , Protéines tumorales/métabolisme , Leucémie-lymphome lymphoblastique à précurseurs T/traitement médicamenteux , Transduction du signal , Réponse aux protéines mal repliées , Animaux , Division cellulaire , Chaperonne BiP du réticulum endoplasmique , Humains , Souris , Souris de lignée NOD , Souris SCID , Protéines tumorales/composition chimique , Phénazines , Leucémie-lymphome lymphoblastique à précurseurs T/anatomopathologieRÉSUMÉ
B-precursor acute lymphoblastic leukemia (B-pre ALL) is a malignant disorder characterized by the abnormal proliferation of B-cell progenitors. The prognosis of B-pre ALL has improved in pediatric patients, but the outcome is much less successful in adults. Constitutive activation of the phosphatidylinositol 3-kinase (PI3K), Akt and the mammalian target of rapamycin (mTOR) (PI3K/Akt/mTOR) network is a feature of B-pre ALL, where it strongly influences cell growth and survival. RAD001, a selective mTORC1 inhibitor, has been shown to be cytotoxic against many types of cancer including hematological malignancies. To investigate whether mTORC1 could represent a target in the therapy of B-pre ALL, we treated cell lines and adult patient primary cells with RAD001. We documented that RAD001 decreased cell viability, induced cell cycle arrest in G0/G1 phase and caused apoptosis in B-pre ALL cell lines. Autophagy was also induced, which was important for the RAD001 cytotoxic effect, as downregulation of Beclin-1 reduced drug cytotoxicity. RAD001 strongly synergized with the novel allosteric Akt inhibitor MK-2206 in both cell lines and patient samples. Similar results were obtained with the combination CCI-779 plus GSK 690693. These findings point out that mTORC1 inhibitors, either as a single agent or in combination with Akt inhibitors, could represent a potential therapeutic innovative strategy in B-pre ALL.
Sujet(s)
Inhibiteurs des phosphoinositide-3 kinases , Leucémie-lymphome lymphoblastique à précurseurs B/traitement médicamenteux , Protéines proto-oncogènes c-akt/antagonistes et inhibiteurs , Transduction du signal/effets des médicaments et des substances chimiques , Sérine-thréonine kinases TOR/antagonistes et inhibiteurs , Apoptose/effets des médicaments et des substances chimiques , Autophagie/effets des médicaments et des substances chimiques , Points de contrôle du cycle cellulaire/effets des médicaments et des substances chimiques , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Évérolimus , Composés hétérocycliques 3 noyaux/pharmacologie , Humains , Complexe-1 cible mécanistique de la rapamycine , Complexes multiprotéiques/antagonistes et inhibiteurs , Oxadiazoles/pharmacologie , Sirolimus/analogues et dérivés , Sirolimus/pharmacologieRÉSUMÉ
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G(0)/G(1) phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3α/ß and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34(+)/CD4(-)/CD7(-)), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL.
Sujet(s)
Composés hétérocycliques 3 noyaux/pharmacologie , Leucémie-lymphome lymphoblastique à précurseurs T/anatomopathologie , Inhibiteurs de protéines kinases/pharmacologie , Protéines proto-oncogènes c-akt/antagonistes et inhibiteurs , Antinéoplasiques/pharmacologie , Apoptose/effets des médicaments et des substances chimiques , Autophagie/effets des médicaments et des substances chimiques , Technique de Western , Cycle cellulaire/effets des médicaments et des substances chimiques , Doxorubicine/pharmacologie , Synergie des médicaments , Humains , Phosphorylation , Leucémie-lymphome lymphoblastique à précurseurs T/enzymologie , Transduction du signalRÉSUMÉ
The mammalian target of rapamycin (mTOR) serine/threonine kinase is the catalytic subunit of two multi-protein complexes, referred to as mTORC1 and mTORC2. Signaling downstream of mTORC1 has a critical role in leukemic cell biology by controlling mRNA translation of genes involved in both cell survival and proliferation. mTORC1 activity can be downmodulated by upregulating the liver kinase B1/AMP-activated protein kinase (LKB1/AMPK) pathway. Here, we have explored the therapeutic potential of the anti-diabetic drug, metformin (an LKB1/AMPK activator), against both T-cell acute lymphoblastic leukemia (T-ALL) cell lines and primary samples from T-ALL patients displaying mTORC1 activation. Metformin affected T-ALL cell viability by inducing autophagy and apoptosis. However, it was much less toxic against proliferating CD4(+) T-lymphocytes from healthy donors. Western blot analysis demonstrated dephosphorylation of mTORC1 downstream targets. Unlike rapamycin, we found a marked inhibition of mRNA translation in T-ALL cells treated with metformin. Remarkably, metformin targeted the side population of T-ALL cell lines as well as a putative leukemia-initiating cell subpopulation (CD34(+)/CD7(-)/CD4(-)) in patient samples. In conclusion, metformin displayed a remarkable anti-leukemic activity, which emphasizes future development of LKB1/AMPK activators as clinical candidates for therapy in T-ALL.
Sujet(s)
Adenylate kinase/métabolisme , Leucémie-lymphome lymphoblastique à précurseurs T/métabolisme , Protéines/métabolisme , Transduction du signal , Apoptose , Séquence nucléotidique , Lignée cellulaire tumorale , Amorces ADN , Cytométrie en flux , Humains , Complexe-1 cible mécanistique de la rapamycine , Metformine/pharmacologie , Complexes multiprotéiques , Phosphorylation , Leucémie-lymphome lymphoblastique à précurseurs T/anatomopathologie , Biosynthèse des protéines/effets des médicaments et des substances chimiques , ARN messager/génétique , Réaction de polymérisation en chaine en temps réel , Sérine-thréonine kinases TORRÉSUMÉ
The mammalian Target Of Rapamycin (mTOR) serine/threonine kinase belongs to two multi-protein complexes, referred to as mTORC1 and mTORC2. mTOR-generated signals have critical roles in leukemic cell biology by controlling mRNA translation of genes that promote proliferation and survival. However, allosteric inhibition of mTORC1 by rapamycin has only modest effects in T-cell acute lymphoblastic leukemia (T-ALL). Recently, ATP-competitive inhibitors specific for the mTOR kinase active site have been developed. In this study, we have explored the therapeutic potential of active-site mTOR inhibitors against both T-ALL cell lines and primary samples from T-ALL patients displaying activation of mTORC1 and mTORC2. The inhibitors affected T-ALL cell viability by inducing cell-cycle arrest in G(0)/G(1) phase, apoptosis and autophagy. Western blot analysis demonstrated a Ser 473 Akt dephosphorylation (indicative of mTORC2 inhibition) and a dephosphorylation of mTORC1 downstream targets. Unlike rapamycin, we found a marked inhibition of mRNA translation in T-ALL cell lines treated with active-site mTOR inhibitors. The inhibitors strongly synergized with both vincristine and the Bcl-2 inhibitor, ABT-263. Remarkably, the drugs targeted a putative leukemia-initiating cell sub-population (CD34(+)/CD7(-)/CD4(-)) in patient samples. In conclusion, the inhibitors displayed remarkable anti-leukemic activity, which emphasizes their future development as clinical candidates for therapy in T-ALL.
Sujet(s)
Apoptose/effets des médicaments et des substances chimiques , Autophagie/effets des médicaments et des substances chimiques , Leucémie-lymphome lymphoblastique à précurseurs T/traitement médicamenteux , Leucémie-lymphome lymphoblastique à précurseurs T/anatomopathologie , Inhibiteurs de protéines kinases/pharmacologie , Protéines/antagonistes et inhibiteurs , Facteurs de transcription/antagonistes et inhibiteurs , Animaux , Technique de Western , Domaine catalytique , Cycle cellulaire/effets des médicaments et des substances chimiques , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Cytométrie en flux , Humains , Immunosuppresseurs/pharmacologie , Complexe-1 cible mécanistique de la rapamycine , Souris , Complexes multiprotéiques , Phosphorylation/effets des médicaments et des substances chimiques , Leucémie-lymphome lymphoblastique à précurseurs T/métabolisme , Protéines/métabolisme , ARN messager/génétique , RT-PCR , Sirolimus/pharmacologie , Sérine-thréonine kinases TOR , Facteurs de transcription/métabolismeRÉSUMÉ
There exists an active lipid metabolism in the nucleus, which is regulated differentially from the lipid metabolism taking place elsewhere in the cell. Evidence has been accumulated that nuclear lipid metabolism is closely involved in a variety of cell responses, including proliferation, differentiation, and apoptosis. A fundamental lipid second messenger which is generated in the nucleus is diacylglycerol, that is mainly known for its role as an activator of some protein kinase C isoforms. Diacylglycerol kinases attenuate diacylglycerol signaling by converting this lipid to phosphatidic acid, which also has signaling functions. Ten mammalian diacylglycerol kinase isoforms have been cloned so far, and some of them are found also in the nucleus, either as resident proteins or after migration from cytoplasm in response to various agonists. Experiments using cultured cells have demonstrated that nuclear diacylglycerol kinases have prominent roles in cell cycle regulation and differentiation. In this review, the emerging roles played by diacylglycerol kinases in the nucleus, such as the control of G1/S phase transition, are discussed.
Sujet(s)
Noyau de la cellule/enzymologie , Diacylglycérol kinase/métabolisme , Transduction du signal , Animaux , Différenciation cellulaire/physiologie , Prolifération cellulaire , Humains , Isoenzymes/métabolisme , Métabolisme lipidiqueRÉSUMÉ
Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) have been implicated in the pathophysiology of many human cancers, including those of hematopoietic lineage. We investigated the therapeutic potential of the novel IGF-IR tyrosine kinase activity inhibitor, NVP-AEW541, on human acute myeloid leukemia (AML) cells. NVP-AEW541 was tested on a HL60 cell subclone, which is dependent on autocrine secretion of IGF-I for survival and drug resistance, as well as primary drug resistant leukemia cells. NVP-AEW541 treatment (24 h) induced dephosphorylation of IGF-IR. NVP-AEW541 also caused Akt dephosphorylation and changes in the expression of key regulatory proteins of the cell cycle. At longer incubation times (48 h), NVP-AEW541-induced apoptotic cell death, as demonstrated by caspase-3 cleavage. Apoptosis was accompanied by decreased expression of anti-apoptotic proteins. NVP-AEW541 enhanced sensitivity of HL60 cells to either cytarabine or etoposide. Moreover, NVP-AEW541 reduced the clonogenic capacity of AML CD34(+) cells cultured in the presence of IGF-I. Chemoresistant AML blasts displayed enhanced IGF-I secretion, and were sensitized to etoposide-induced apoptosis by NVP-AEW541. Our findings indicate that NVP-AEW541 might be a promising therapeutic agent for the treatment of those AML cases characterized by IGF-I autocrine secretion.
Sujet(s)
Apoptose/effets des médicaments et des substances chimiques , Facteur de croissance IGF-I/métabolisme , Leucémie aigüe myéloïde/traitement médicamenteux , Pyrimidines/pharmacologie , Pyrroles/pharmacologie , Récepteur IGF de type 1/antagonistes et inhibiteurs , Inhibiteur p27 de kinase cycline-dépendante , Cytarabine/pharmacologie , Régulation négative , Étoposide/pharmacologie , Cellules HL-60 , Humains , Protéines et peptides de signalisation intracellulaire/analyse , Leucémie aigüe myéloïde/métabolisme , Leucémie aigüe myéloïde/anatomopathologie , Inhibiteurs des phosphoinositide-3 kinases , Phosphorylation , Récepteur IGF de type 1/métabolismeRÉSUMÉ
The existence of intranuclear lipid-dependent signal transduction systems has been demonstrated by several independent groups. Remarkably, intranuclear lipid-dependent signal transduction pathways are regulated independently from their membrane/cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation, differentiation, and apoptosis. Diacylglycerol (DG) is a fundamental lipid second messenger which is produced in the nucleus. Since the levels of nuclear DG fluctuate during the cell cycle progression, it has been suggested that this lipid second messenger has important regulatory roles. Most likely, nuclear DG serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases (DGKs), i.e. the enzymes that, by converting DG into phosphatidic acid (PA), terminate DG-dependent events. This review aims at highlighting the different isozymes of DGKs present within the nucleus as well as at discussing their potential functions with particular emphasis placed on DNA replication.
Sujet(s)
Noyau de la cellule/enzymologie , Réplication de l'ADN , Diacylglycérol kinase/biosynthèse , Animaux , Noyau de la cellule/génétique , Diacylglycérol kinase/génétique , Régulation de l'expression des gènes codant pour des enzymes , Humains , Isoenzymes/biosynthèse , Isoenzymes/génétique , Métabolisme lipidique/génétique , Souris , RatsRÉSUMÉ
The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is crucial to many aspects of cell growth, survival and apoptosis, and its constitutive activation has been implicated in the both the pathogenesis and the progression of a wide variety of neoplasias. Hence, this pathway is an attractive target for the development of novel anticancer strategies. Recent studies showed that PI3K/Akt signaling is frequently activated in acute myeloid leukemia (AML) patient blasts and strongly contributes to proliferation, survival and drug resistance of these cells. Upregulation of the PI3K/Akt network in AML may be due to several reasons, including FLT3, Ras or c-Kit mutations. Small molecules designed to selectively target key components of this signal transduction cascade induce apoptosis and/or markedly increase conventional drug sensitivity of AML blasts in vitro. Thus, inhibitory molecules are currently being developed for clinical use either as single agents or in combination with conventional therapies. However, the PI3K/Akt pathway is important for many physiological cellular functions and, in particular, for insulin signaling, so that its blockade in vivo might cause severe systemic side effects. In this review, we summarize the existing knowledge about PI3K/Akt signaling in AML cells and we examine the rationale for targeting this fundamental signal transduction network by means of selective pharmacological inhibitors.
Sujet(s)
Leucémie myéloïde/métabolisme , Phosphatidylinositol 3-kinases/métabolisme , Protéines proto-oncogènes c-akt/métabolisme , Transduction du signal , Maladie aigüe , Humains , Leucémie myéloïde/traitement médicamenteux , Modèles biologiques , Inhibiteurs des phosphoinositide-3 kinases , Inhibiteurs de protéines kinases/pharmacologie , Inhibiteurs de protéines kinases/usage thérapeutique , Protéines proto-oncogènes c-akt/antagonistes et inhibiteurs , Transduction du signal/effets des médicaments et des substances chimiquesRÉSUMÉ
Several studies have demonstrated the existence of an autonomous intranuclear phospho-inositide cycle that involves the activation of nuclear PI-PLC and the generation of diacylglycerol (DG) within the nucleus. Although several distinct isozymes of PI-PLC have been detected in the nucleus, the isoform that has been most consistently highlighted as being nuclear is PI-PLC-beta1. Nuclear PI-PLC-beta1 has been linked with either cell proliferation or differentiation. Remarkably, the activation mechanism of nuclear PI-PLC-beta1 has been shown to be different from its plasma membrane counterpart, being dependent on phosphorylation effected by p44/42 mitogen activated protein (MAP) kinase. In this review, we report the most up-dated findings about nuclear PI-PLC-beta1, such as the localization in nuclear speckles, the activity changes during the cell cycle phases, and the possible involvement in the progression of myelodisplastic syndrome to acute myeloid leukemia.
Sujet(s)
Noyau de la cellule/enzymologie , Isoenzymes/physiologie , Lipides/physiologie , Transduction du signal/physiologie , Type C Phospholipases/physiologie , Séquence d'acides aminés , Animaux , Cycle cellulaire/physiologie , Noyau de la cellule/composition chimique , Humains , Isoenzymes/composition chimique , Isoenzymes/génétique , Données de séquences moléculaires , Protéines tumorales/composition chimique , Protéines tumorales/génétique , Protéines tumorales/physiologie , Phospholipase C beta , Type C Phospholipases/composition chimique , Type C Phospholipases/génétiqueRÉSUMÉ
A major factor undermining successful cancer treatment is the occurrence of resistance to conventional treatments such as chemotherapy and ionizing radiation. Evidence accumulated over the recent years has indicated the phosphoinositide 3-kinase/Akt signal transduction pathway as one of the major factors implicated in cancer resistance to conventional therapies. Indeed, the phosphoinositide 3-kinase/Akt axis regulates the expression and/or function of many anti-apoptotic proteins which strongly contributes to cancer cell survival. As a result, small molecules designed to specifically target key components of this signaling network are now being developed for clinical use as single therapeutic agents and/or in combination with other forms of therapy to overcome resistance. Initially, the phosphoinositide 3-kinase/Akt signal transduction pathway has been mainly investigated in solid tumors. Recently, however, this network has also been recognized as an important therapeutic target in human leukemias. Specific inhibition of this signalling pathway may be a valid approach to treat these diseases and increase the efficacy of standard types of therapy.
Sujet(s)
Résistance aux médicaments antinéoplasiques/physiologie , Leucémies/enzymologie , Phosphatidylinositol 3-kinases/physiologie , Protein-Serine-Threonine Kinases/physiologie , Protéines proto-oncogènes/physiologie , Humains , Isoenzymes/physiologie , Protein kinases/physiologie , Protein-Serine-Threonine Kinases/antagonistes et inhibiteurs , Protein-Serine-Threonine Kinases/génétique , Protéines proto-oncogènes/antagonistes et inhibiteurs , Protéines proto-oncogènes/génétique , Protéines proto-oncogènes c-akt , Transduction du signal/physiologie , Sérine-thréonine kinases TORRÉSUMÉ
It is now well established that the reduced capacity of tumor cells of undergoing cell death through apoptosis plays a key role both in the pathogenesis of cancer and in therapeutic treatment failure. Indeed, tumor cells frequently display multiple alterations in signal transduction pathways leading to either cell survival or apoptosis. In mammals, the pathway based on phosphoinositide 3-kinase (PI3K)/Akt conveys survival signals of extreme importance and its downregulation, by means of pharmacological inhibitors of PI3K, considerably lowers resistance to various types of therapy in solid tumors. We recently described an HL60 leukemia cell clone (HL60AR cells) with a constitutively active PI3K/Akt pathway. These cells were resistant to multiple chemotherapeutic drugs, all-trans-retinoic acid (ATRA), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Treatment with two pharmacological inhibitors of PI3K, wortmannin and Ly294002, restored sensitivity of HL60AR cells to the aforementioned treatments. However, these inhibitors have some drawbacks that may severely limit or impede their clinical use. Here, we have tested whether or not a new selective Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (Akt inhibitor), was as effective as Ly294002 in lowering the sensitivity threshold of HL60 cells to chemotherapeutic drugs, TRAIL, ATRA, and ionizing radiation. Our findings demonstrate that, at a concentration which does not affect PI3K activity, the Akt inhibitor markedly reduced resistance of HL60AR cells to etoposide, cytarabine, TRAIL, ATRA, and ionizing radiation. This effect was likely achieved through downregulation of expression of antiapoptotic proteins such as c-IAP1, c-IAP2, cFLIP(L), and of Bad phosphorylation on Ser 136. The Akt inhibitor did not influence PTEN activity. At variance with Ly294002, the Akt inhibitor did not negatively affect phosphorylation of protein kinase C-zeta and it was less effective in downregulating p70S6 kinase (p70S6K) activity. The Akt inhibitor increased sensitivity to apoptotic inducers of K562 and U937, but not of MOLT-4, leukemia cells. Overall, our results indicate that selective Akt pharmacological inhibitors might be used in the future for enhancing the sensitivity of leukemia cells to therapeutic treatments that induce apoptosis or for overcoming resistance to these treatments.
Sujet(s)
Antinéoplasiques/pharmacologie , Résistance aux médicaments antinéoplasiques , Inositol/pharmacologie , Protéines et peptides de signalisation intracellulaire , Protein-Serine-Threonine Kinases , Protéines proto-oncogènes/antagonistes et inhibiteurs , Trétinoïne/pharmacologie , Technique de Western , Protéine de régulation de l'apoptose CASP8 et FADD-like , Protéines de transport/métabolisme , Caspases/métabolisme , 4H-1-Benzopyran-4-ones/pharmacologie , Cytarabine/pharmacologie , Cytochromes de type c/métabolisme , Antienzymes/pharmacologie , Étoposide/pharmacologie , Cellules HL-60/effets des médicaments et des substances chimiques , Cellules HL-60/effets des radiations , Humains , Protéines IAP , Inositol/analogues et dérivés , Isoenzymes/antagonistes et inhibiteurs , Isoenzymes/métabolisme , Morpholines/pharmacologie , Phosphohydrolase PTEN , Phosphatidylinositol 3-kinases/métabolisme , Inhibiteurs des phosphoinositide-3 kinases , Phosphoric monoester hydrolases/métabolisme , Phosphorylation , Protéine kinase C/antagonistes et inhibiteurs , Protéine kinase C/métabolisme , Protein Kinase C-theta , Protéines/métabolisme , Protéines proto-oncogènes/métabolisme , Protéines proto-oncogènes c-akt , Rayonnement ionisant , Ribosomal Protein S6 Kinases, 70-kDa/métabolisme , Transfection , Protéines suppresseurs de tumeurs/métabolisme , Ubiquitin-protein ligases , Protéine BadRÉSUMÉ
The serine/threonine protein kinase Akt, a downstream effector of phosphoinositide 3-kinase (PI3K), plays a pivotal role in tumorigenesis because it affects the growth and survival of cancer cells. Several laboratories have demonstrated that Akt inhibits transcriptional activation of a number of related forkhead transcription factors now referred to as FoxO1, FoxO3, and FoxO4. Akt-regulated forkhead transcription factors are involved in the control of the expression of both the cyclin-dependent kinase (cdk) inhibitor p27(Kip1) and proapoptotic Bim protein. Very little information is available concerning the importance of the PI3K/Akt pathway in HL60 human leukemia cells. Here, we present our findings showing that the PI3K/Akt axis regulates cell cycle progression of HL60 cells through multiple mechanisms also involving the control of FoxO1 and FoxO3. To this end, we took advantage of a HL60 cell clone (HL60AR cells) with a constitutively activated PI3K/Akt axis. When compared with parental (PT) HL60 cells, HL60AR cells displayed higher levels of phosphorylated FoxO1 and FoxO3. In AR cells forkhead factors localized predominantly in the cytoplasm, whereas in PT cells they were mostly nuclear. AR cells proliferated faster than PT cells and showed a lower amount of the cdk inhibitor p27(Kip1), which was mainly found in the cytoplasm and was hyperphosphorylated on threonine residues. AR cells also displayed higher levels of cyclin D1 and phosphorylated p110 Retinoblastoma protein. The protein levels of cdk2, cdk4, and cdk6 were not altered in HL60AR cells, whereas the activities of both ckd2 and cdk6 were higher in AR than in PT cells. These results show that in HL60 cells the PI3K/Akt signaling pathway may be involved in the control of the cell cycle progression most likely through mechanisms involving the activation of forkhead transcription factors.
Sujet(s)
Protéines du cycle cellulaire/métabolisme , Cycle cellulaire/physiologie , Cycline D1/génétique , Phosphatidylinositol 3-kinases/métabolisme , Protein-Serine-Threonine Kinases , Protéines proto-oncogènes/métabolisme , Protéines suppresseurs de tumeurs/métabolisme , Androstadiènes/pharmacologie , Protéines du cycle cellulaire/génétique , Noyau de la cellule/enzymologie , Inhibiteur p27 de kinase cycline-dépendante , Antienzymes/métabolisme , Antienzymes/pharmacologie , Technique d'immunofluorescence , Phase G1/physiologie , Régulation de l'expression des gènes tumoraux , Cellules HL-60 , Humains , Protéines proto-oncogènes c-akt , Facteurs de transcription/métabolisme , Protéines suppresseurs de tumeurs/génétique , WortmannineRÉSUMÉ
TRAIL is a member of the tumor necrosis factor superfamily which induces apoptosis in cancer but not in normal cells. Akt1 promotes cell survival and blocks apoptosis. The scope of this paper was to investigate whether a HL60 human leukemia cell clone (named AR) with constitutively active Akt1 was resistant to TRAIL. We found that parental (PT) HL60 cells were very sensitive to a 6 h incubation in the presence of TRAIL and died by apoptosis. In contrast, AR cells were resistant to TRAIL concentrations as high as 2 microg/ml for 24 h. Two pharmacological inhibitors of PI3K, Ly294002 and wortmannin, restored TRAIL sensitivity of AR cells. AR cells stably overexpressing PTEN had lower Akt1 activity and were sensitive to TRAIL. Conversely, PT cells stably overexpressing a constitutive active form of Akt1 became TRAIL resistant. TRAIL activated caspase-8 but not caspase-9 or -10 in HL60 cells. We did not observe a protective effect of Bcl-X(L) or Bcl-2 against the cytotoxic activity of TRAIL, even though TRAIL induced cleavage of BID. There was a close correlation between TRAIL sensitivity and intranuclear presence of the p50 subunit of NF-kappaB. Higher levels of the FLICE inhibitory protein, cFLIP(L), were observed in TRAIL-resistant cells. Both the cell permeable NF-kappaB inhibitor SN50 and cycloheximide lowered cFLIP(L)expression and restored sentivity of AR cells to TRAIL. Our results suggest that Akt1 may be an important regulator of TRAIL sensitivity in HL60 cells through the activation of NF-kappaB and up-regulation of cFLIP(L) synthesis.
Sujet(s)
Apoptose/effets des médicaments et des substances chimiques , Protéines de transport/métabolisme , Protéines et peptides de signalisation intracellulaire , Glycoprotéines membranaires/toxicité , Facteur de transcription NF-kappa B/métabolisme , Protein-Serine-Threonine Kinases/métabolisme , Protéines proto-oncogènes , Facteur de nécrose tumorale alpha/toxicité , Protéines régulatrices de l'apoptose , Protéine de régulation de l'apoptose CASP8 et FADD-like , 4H-1-Benzopyran-4-ones/pharmacologie , Cytosol/effets des médicaments et des substances chimiques , Cytosol/physiologie , Résistance aux médicaments antinéoplasiques , Antienzymes/pharmacologie , Régulation de l'expression des gènes tumoraux/effets des médicaments et des substances chimiques , Cellules HL-60 , Humains , Glycoprotéines membranaires/pharmacocinétique , Mitochondries/effets des médicaments et des substances chimiques , Mitochondries/physiologie , Morpholines/pharmacologie , Inhibiteurs des phosphoinositide-3 kinases , Protéines proto-oncogènes c-akt , Ligand TRAIL , Facteur de nécrose tumorale alpha/pharmacocinétiqueRÉSUMÉ
It is becoming increasingly evident that stimulation of nuclear lipid metabolism plays a central role in many signal transduction pathways that ultimately result in various cell responses including proliferation and differentiation. Nuclear lipid metabolism seems to be at least as complex as that existing at the plasma membrane. However, a distinctive feature of nuclear lipid biochemical pathways is their operational independence from their cell periphery counterparts. Although initially it was thought that nuclear lipids would serve as a source for second messengers, recent evidence points to the likelihood that lipids present in the nucleus also fulfil other roles. The aim of this review is to highlight the most intriguing advances made in the field over the last year, such as the production of new probes for the in situ mapping of nuclear phosphoinositides, the identification of two sources for nuclear diacylglycerol production, the emerging details about the peculiar regulation of nuclear phosphoinositide synthesizing enzymes, and the distinct possibility that nuclear lipids are involved in processes such as chromatin organization and pre-mRNA splicing.
Sujet(s)
Noyau de la cellule/composition chimique , Noyau de la cellule/métabolisme , Métabolisme lipidique , Diglycéride/métabolisme , Humains , Sondes moléculaires/métabolisme , Phosphatidyl inositols/métabolisme , Transduction du signal/physiologieRÉSUMÉ
The nuclear matrix is defined as the residual framework after the removal of the nuclear envelope, chromatin, and soluble components by sequential extractions. According to several investigators the nuclear matrix provides the structural basis for intranuclear order. However, the existence itself and the nature of this structure is still uncertain. Although the techniques used for the visualization of the nuclear matrix have improved over the years, it is still unclear to what extent the isolated nuclear matrix corresponds to an in vivo existing structure. Therefore, considerable skepticism continues to surround the nuclear matrix fraction as an accurate representation of the situation in living cells. Here, we summarize the experimental evidence in favor of, or against, the presence of a diffuse nucleoskeleton as a facilitating organizational nonchromatin structure of the nucleus.
Sujet(s)
Noyau de la cellule/physiologie , Matrice nucléaire/physiologie , Animaux , Noyau de la cellule/métabolisme , Noyau de la cellule/ultrastructure , Humains , Matrice nucléaire/métabolisme , Matrice nucléaire/ultrastructure , Protéines nucléaires/métabolisme , Fixation tissulaireRÉSUMÉ
Several independent groups have shown that lipid-dependent signal transduction systems operate in the nucleus and that they are regulated independently from their membrane and cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation and differentiation. Diacylglycerol is a fundamental lipid second messenger which is produced in the nucleus. The levels of nuclear diacylglycerol fluctuate during the cell cycle progression, suggesting that such a molecule has important regulatory roles. Most likely, nuclear diacylglycerol serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases, i.e. the enzymes that, by converting diacylglycerol into phosphatidic acid, terminate diacylglycerol-dependent events. A number of diacylglycerol kinases encoded by separate genes are present in the mammalian genome. This review aims at highlighting the different isotypes of diacylglycerol kinases identified at the nuclear level as well as at discussing their potential function and regulation.
Sujet(s)
Noyau de la cellule/enzymologie , Diacylglycérol kinase/physiologie , Animaux , Noyau de la cellule/métabolisme , Humains , Lipides/physiologie , Souris , Modèles biologiques , Isoformes de protéines/physiologie , Rats , Transduction du signalRÉSUMÉ
Recent reports have highlighted that phosphoinositide-specific phospholipase Cbeta1 expression is linked to neuronal differentiation in different experimental models. We sought to determine whether or not this is also true for nerve growth factor (NGF)-induced neuronal differentiation of rat PC12 cells. However, we did not find differences in the expression of both the forms of phosphoinositide-specific phospholipase Cbeta1 (a and b) during sympathetic differentiation of these cells. Also, PC12 cell clones stably overexpressing phosphoinositide-specific phospholipase Cbeta1 were not more susceptible to the differentiating effect of NGF. Furthermore, since it is well established that phosphoinositide-specific phospholipase Cbeta1 affects cell proliferation, we investigated whether or not PC12 cell clones stably overexpressing phosphoinositide-specific phospholipase Cbeta1 showed differences in survival to serum deprivation and cell cycle, when compared to wild type cells. Nevertheless, we did not find any differences in these parameters between wild type cells and the overexpressing clones. Interestingly, in PC12 cells the overexpressed phosphoinositide-specific phospholipase Cbeta1 did not localize to the nucleus, but by immunofluorescence analysis, was detected in the cytoplasm. Therefore, our findings may represent another important clue to the fact that only when it is located within the nucleus phosphoinositide-specific phospholipase Cbeta1 is able to influence cell proliferation.
Sujet(s)
Différenciation cellulaire/effets des médicaments et des substances chimiques , Isoenzymes/métabolisme , Facteur de croissance nerveuse/pharmacologie , Cellules PC12/effets des médicaments et des substances chimiques , Phosphatidyl inositols/métabolisme , Type C Phospholipases/métabolisme , Animaux , Apoptose/physiologie , Cycle cellulaire/effets des médicaments et des substances chimiques , Cycle cellulaire/physiologie , Différenciation cellulaire/physiologie , Survie cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/physiologie , Milieux de culture sans sérum/métabolisme , Cytoplasme/métabolisme , Hydrolyse , Cellules PC12/enzymologie , Cellules PC12/anatomopathologie , Phospholipase C beta , RatsRÉSUMÉ
Although inositol lipids constitute only a very minor proportion of total cellular lipids, they have received immense attention by scientists since it was discovered that they play key roles in a wide range of important cellular processes. In the late 1980s, it was suggested that these lipids are also present within the cell nucleus. Albeit the early reports about the intranuclear localization of phosphoinositides were met by skepticism and disbelief, compelling evidence has subsequently been accumulated convincingly showing that a phosphoinositide cycle is present at the nuclear level and may be activated in response to stimuli that do not activate the inositol lipid metabolism localized at the plasma membrane. Very recently, intriguing new data have highlighted that some of the mechanisms regulating nuclear inositol lipid metabolism differ in a substantial way from those operating at the cell periphery. Here, we provide an overview of recent findings regarding the regulation of both nuclear phosphatidylinositol 3-kinase and phosphoinositide-specific phospholipase C-beta1.
Sujet(s)
Noyau de la cellule/métabolisme , Inositol/métabolisme , Isoenzymes/métabolisme , Métabolisme lipidique , Phosphatidylinositol 3-kinases/métabolisme , Type C Phospholipases/métabolisme , Animaux , Humains , Phosphatidyl inositols/métabolisme , Phospholipase C beta , PhosphorylationRÉSUMÉ
Apoptosis is a form of active cell death essential for morphogenesis, development, differentiation, and homeostasis of multicellular organisms. The activation of genetically controlled specific pathways that are highly conserved during evolution results in the characteristic morphological features of apoptosis that are mainly evident in the nucleus. These include chromatin condensation, nuclear shrinkage, and the formation of apoptotic bodies. The morphological changes are the result of molecular alterations, such as DNA and RNA cleavage, post-translational modifications of nuclear proteins, and proteolysis of several polypeptides residing in the nucleus. During the last five years our understanding of the process of apoptosis has dramatically increased. However, the mechanisms that lead to apoptotic changes in the nucleus have been only partially clarified. Here, we shall review the most recent findings that may explain why the nucleus displays these striking modifications. Moreover, we shall take into consideration the emerging evidence about apoptotic events as a trigger for the generation of autoantibodies to nuclear components.