1,721,063 research outputs found
Characterization and role of the transcription factor cAMP response element binding protein (CREB) in childhood leukemia
No full textWe investigated the transcription factor cyclic adenosine monophosphate response-element binding protein (CREB) in childhood acute leukemia. We analyzed CREB expression in series of bone marrow samples from children with acute lymphoblast (ALL) or myeloid leukemia (AML) at diagnosis and during remission therapy. We documented that CREB protein was significantly (p<0.001) over expressed in 84% of patients with ALL and 66% of those with AML. CREB protein was always present in the active phosphorylated form, and transcriptional active wholly at diagnosis of leukemia. In order to find the causes of CREB abnormalities we looked at CREB mRNA which was not amplified and at gene sequencing analyses that revealed a wild type genome in leukemic samples. cAMP concentration was then measured but not significant differences in diagnosis samples respect to controls were found. We described an inverse correlation between CREB and its inducible cAMP early repressor (ICER) mRNA expression and decided to considere this phenomenon to be better elucidated. ICER and CREB are both transcriptional activator and repressor, respectively, of the cAMP-mediated signaling pathway. The research continued by looking at the possible function of restored ICER expression in leukemia cell lines. ICER exogenously expressed in HL60 was demonstrated to decrease CREB protein level and induce a lowered clonogenic potential in vitro. ICER was demonstrated to repress a series of genes involved in cell cycle, proliferation and MAPK signaling. Its binding to CRE promoters confirmed its role in controlling gene expression of the main cellular pathways. HL60 cell line expressing ICER was then evaluated in their ability to repopulate non-obese diabetic-severe combined immunodeficient mice demonstrating a lower extramedullary sites invasion and bone marrow angiogenesis. So, its potential effects on tumor progression was documented in vivo. We then tried to give a reason of ICER lower expression found in HL60 and demonstrated that it was subjected to degradation through a constitutively active form of the extracellular signal-regulated protein kinase (ERK1/2) maintained by CREB, which drives it to the proteasome. Considerable attention was focused on the role played by ICER in the control of genes related to apoptosis and MAPK signaling and chemotherapics were used as stimuli for cell death. We found that a series of ICER’s target cyclins, phosphatases DUSP1/4, kinases and oncogenes were strongly downregulated after chemotherapic treatment, supporting ICER restored expression to confer an enhanced sensibility to drugs to leukemic cells. Cell cycle was impaired and apoptosis significantly increased. Addressing the mechanism by which exogenous ICER improves apoptosis, we found that the simple inhibition of DUSP1/4 phosphatases expression triggers the activation of the p38 apoptotic pathway and the disruption of ERK, AKT and RAS survival signaling. This is a process caspases dependent and abrogated by p38 specific inhibitor. We reveal that ICER induces CREB proteasome degradation supporting apoptotic signaling and a lowered tumorigenicity. ICER is here proposed to be able to switch leukemia cell fate from survival to apoptosis by counteracting CREB transcriptional activity and modifing its protein stability.
At this point, we revealed that CREB was overexpressed in leukemia, but the underlying mechanism remained unknown. MicroRNAs , that act as negative regulators of gene expression principally through translational repression, were then investigated for the mediation of high CREB protein levels. A series of miRNAs which target CREB were identified. RQ-PCR revealed that miR-34b was expressed significantly less in myeloid cell lines, previously known for high CREB protein levels. Exogenous miR-34b expression was induced, and results revealed a direct interaction with CREB in vitro. MiR-34b restored expression caused cell cycle abnormalities, reduced anchorage independent growth, and altered CREB target gene expression, suggesting its suppressor potential. CREB target proteins (BCL-2, Cyclins A1, B1, D, NfKB, JAK1, STAT3), as well as many downstream protein kinases and cell survival signaling pathways (AKT/mTOR, ERK) usually elicited by CREB, were observed to have decreased. The miR-34b/34c promoter was demonstrated to be methylated finding this epigenetic regulation able to control the observed lower miR-34b expression levels in order to maintain the CREB protein overexpressed. In addition, the inverse correlation between miR-34b and CREB expression was found in a cohort of pediatric patients at diagnosis of acute myeloid leukemia, supporting this relationship also in vivo.
Finally, the research defines CREB as an oncogene and ICER as a tumor suppressor in leukemia. Unbalanced CREB/ICER expression needs to be considered a pathogenetic feature in leukemogenesis. Our results identify a targets and pathways to be considered for future therapeutic approaches. The direct miR-34b targeting of CREB provides new information about myeloid transformation.In questo dottorato di ricerca è stato studiato il fattore di trascrizione CREB che dirige l’espressione genica regolata per lo più dall’ AMPciclico. CREB è uno dei fattori di trascrizione più conosciuti, ma ancora poco è noto del suo ruolo fisiologico e oncologico nel tessuto ematopoietico. L’espressione proteica di CREB è stata definita in linee cellulari leucemiche e in una ampia coorte di pazienti all’esordio di leucemia acuta linfoide (LLA) o leucemia acuta mieloide (LMA). I dati sono stati confrontati con quelli ottenuti dall’analisi di prelievi raccolti durante il follow up in documentata remissione di malattia e donatori sani. I risultati hanno evidenziato una elevata espressione proteica di CREB nell’84% delle ALL e nel 66% delle LAM (p<0.001). Il legame specifico di CREB con i siti di riconoscimento cAMP-Responsive-Element (CRE, 5’TGACGTCA3’) sui promotori si è dimostrato avvenire esclusivamente nei prelievi alla diagnosi di leucemia dove dunque l’overespressione di CREB sosteneva la possibilità di una sua attività trascrizionale anomala. Per capire le cause di questo fenomeno sono stati misurati i livelli di CREB mRNA che non erano significativamente differenti negli esordi rispetto ai controlli utilizzati. E’ stato condotto il sequenziamento del gene CREB, e dei domini di interazione tra CREB la CREB binding protein (CBP) in quanto necessario alla attivazione trascrizionale, che però non ha rivelato nessuna mutazione tale da giustificare l’overespressione di CREB. Infine è stata misurata la quantità di cAMP che non è risultata significativamente diversa nei campioni all’esordio rispetto ai controlli. Allo stesso fine, è stata valutata l’espressione del cAMP inducible early repressor (ICER). ICER si è dimostrato essere sotto espresso all’esordio di leucemia e upregolato nei prelievi in remissione e nei donatori sani, contrariamenti ai livelli espressione di CREB documentati nei medesimi campioni.
La ricerca è proseguita con lo studio di ICER, che è un fattore di trascrizione noto riconoscere CRE nei promotori con la funzione di reprimerne la trascrizione. ICER dunque ha il ruolo di modulare l’attività di CREB controllando l’espressione genica in diversi tessuti. Essendo ICER sotto espresso nel tessuto leucemico è stato da noi costruito un modello in vitro reintroducendo una esogena espressione di ICER in linee cellulari. E’ stato dimostrato che l’espressione esogena di ICER induceva una diminuzione della proteina CREB e una alterazione di una serie di geni target. I promotori di alcuni geni, scelti per svolgere attività cellulari primarie quali la proliferazione e la sopravvivenza, sono stati dimostrati infatti essere occupati da ICER invece che da CREB. Le attività cellulari delle HL60 risultavano fortemente modificate: la capacità di formare colonie dei blasti esprimenti ICER in vitro era fortemente ridotta, supportando l’ipotesi che ICER potesse svolgere il ruolo di onco-soppressore. E’ stato così utilizzato un test di ripopolamento in vivo in topi NOD-SCID con le HL60 stabilizzate con ICER e HL60 stabilizzate con il vettore vuoto. La linea leucemica si è dimostrata meno tumorigenica se esprimente ICER, la disseminazione dei blasti nel sangue periferico e nella milza era molto rallentata e l’angiogenesi ridotta. L’identificazione di quali geni ICER fosse in grado di reprimere e quindi contrastare il fenotipo leucemico sono stati studiati. I dati di espressione genica hanno dimostrato che ICER reprimeva per lo più geni del ciclo cellulare, dell’apoptosi e del pathway delle MAPK. Con l’utilizzo di chemioterapici come stimoli apoptotici è stato dimostrato che alcune cicline, le fosfatasi DUSP1/4, alcune MAP chinasi e importanti oncogeni, tutte proteine target di ICER, erano fortemente represse nelle cellule leucemiche se esprimenti ICER esogeno, rispetto alle stesse cellule non trattate con farmaci. Il trattamento inoltre induceva un blocco del ciclo cellulare e una aumentata apoptosi dimostrando che ICER conferiva una maggior sensibilità ai farmaci. In particolare,è stato dimostrato che il pathway pro-apoptotico diretto dalla proteina di stress p38 si manteneva attivo grazie alla repressione di DUSP1/4 indotta da ICER, fornendo così il pathway diretto da ICER e nuovi target terapeutici. Infine, sono state indagate le cause della bassa espressione di ICER all’esordio di malattia. E’ stato dimostrato che il pathway legato alla kinase ERK1/2, fortemente mantenuto anche dagli elevati livelli di CREB, permetteva a ERK1/2 di fosforilare ICER e portarlo alla degradazione via proteasoma. Inoltre, è stato dimostrato che una volta reintrodotta l’espressione di ICER in HL60, ICER dimerizzava con CREB portando quest’ultimo alla degradazione via proteasoma, spiegando dunque i bassi livelli di CREB nel nostro modello in vitro.
Infine la ricerca si è concentrata sul cercare di determinare le cause della iper espressione di CREB nella leucemia. L’osservazione che i livelli proteici di CREB erano alterati, ma non i livelli di mRNA, ha condotto lo studio verso il ruolo delle modifiche post-trascrizionali. L’ipotesi che CREB rimanesse ad alti livelli per anomalie di traduzione ha spinto l’analisi verso la ricerca di possibili microRNAs. E’ stato condotto uno studio di 5 micrornas su una serie di linee cellulari mieloidi che ha permesso di puntare su miR-34b in quanto la sua espressione era fortemente abbassata nelle linee leucemiche in concomitanza agli elevati livelli di CREB. MiR-34b è stato così re-inserito nelle linee come oligonucleotide esogeno ed è stato dimostrato che provocava un drastico abbassamento dei livelli di CREB, con conseguente diminuzione dell’espressione genica e proteica dei target di CREB. La vitalità cellulare inoltre era compromessa, compresa la capacità di formare colonie. Il fatto che miR-34b si mantenesse sotto espresso nella leucemia è stato dimostrato dipendere dalla metilazione del suo promotore. Alle analisi in vitro, sono state affiancate le analisi di espressione di miR-34b in una serie di pazienti all’esordio di LAM, dimostrando che anche in vivo miR-34b è fortemente sotttoespresso e CREB upregolato.
Infine, la ricerca ha caratterizzato il gene CREB come un oncogene e ICER come un oncosopressore nella leucemia mieloide. La modulazione della loro espressione risulta di fondamentale importanza nel mantenimento del tumore. La caratterizzazione di nuovi geni target e pathways cellulari implicati nella leucemia mieloide aprono nuove prospettive di studio e di possibilità terapeutiche.La caratterizzazione di CREB come target di miR-34b e la metilazione del promotore come evento regolatore dei livelli di proteina nella leucemia, confermano l’importanza degli eventi epigenetici nella leucemogenesi
Thioridazine derivatives and their use for the treatment of cancer
No full textThe present invention relates to novel thioridazine analog compds. of formula (I): wherein R1 is OCH3 or NH2 , particularly suitable to be used in the treatment of pediatric acute myeloid leukemia harboring the t(6;11)(q27;q23) KMT2A/AFDN rearrangement
BAG-1 IN ACUTE LEUKEMIA: HUNDRED FACES OF A SINGLE PROTEIN
No full textIntroduction. Bcl-2 associated athanogene-1 (Bag-1), a founding member of BAG protein family, is a multifunctional protein which has a role in a wide range of cellular processes including apoptosis, cell survival, transcription, cell motility and proliferation. The involvement of Bag-1 in different cellular pathways can be in part examined by the sub-cellular compartmentalisation of the three major Bag-1 isoforms (Bag-1L, Bag-1M and Bag-1S), generated by alternative translation from a single mRNA, and in part by its interaction with a large number of disparate proteins, including Bcl-2, Raf-1, nuclear hormone receptors, subunits of the ubiquitinylation proteasome complex, Hsc70 and Hsp70. Aims. The elevated level of Bag-1 protein has been confirmed as a considerable index in several malign diseases. To determine significance Bag-1 might have in the processes of leukemogenesis a sequence of human leukemic cell lines and pediatric bone marrow samples with confirmed acute myeloid leukemia were included in our research. Our goal was to clarify the molecular mechanisms potentially in charge for Bag-1 action, in either leukemic cell lines or primary cell cultures. Methods. In vitro studies were based on a small-interfering RNA (siRNA) approach and the results were validated using standard techniques for mRNA and protein expression study. Assays for cell cycle and apoptosis detection were performed. Results. The protein study revealed elevated Bag-1 levels in human leukemic cell lines of both myeloid (ML2, THP1, NOMO1, NB4, MV4;11 and HL60) and lymphoid (REH, RS4;11, 697 and JURKAT) origin. A different expression pattern of Bag-1 protein isoforms was noted for two considered groups of patients, AML or ALL, with changes in protein expression profile at different point of the disease. After Bag-1 was knock-down, a modest effect on cell death or cell cycle profile was observed for the human cell lines while primary cultures showed to be more sensitive to Bag-1 silencing. However, significant decrease was confirmed at the expression level of a wide range of proteins, specially the ones involved in the regulation of apoptosis (Bcl-2, PARP, Caspase-3), cell cycle (p27, CDK2, Cyclin D1) and autophagy (LC3, p62), without affecting the mRNA levels. When double silencing experiments of Bag-1 and Bag-3 (a family co-member) were performed, the effect on cell death and cell cycle arrest were found enforced, suggesting a connection between two proteins to be significant for cells faith in leukemia. Conclusions. Results indicate that the role of Bag-1 in cell death prevention might be more related to lymphoid leukemia, while it might be considered more significant for cell differentiation in myeloid cells. At the same time, elevated Bag-1 protein expression levels in acute leukemia indicates its possible significance for AML and ALL growth. A different expression profile of Bag-1L, Bag-1M and Bag-1S isoforms in myeloid with respect to lymphoid leukemia could lead to the hypothesis that Bag-1 might play a role in leukemia switch, triggering to either ALL or AML phenotype
The cAMP Response Element Binding Protein (CREB) Overexpression Induces Myeloid Transformation in Zebrafish
No full textICER expression inhibits leukemia phenotype and controls tumor progression.
No full textThe inducible cyclic AMP (cAMP) early repressor (ICER) and cAMP response element-binding protein (CREB) are transcriptional regulators of the cAMP-mediated signaling pathway. CREB has been demonstrated to be upregulated in the majority of childhood leukemias contributing to disease progression, whereas ICER, its endogenous repressor, was found to be downregulated. Our research focus has been the function of restored ICER expression. ICER exogenously expressed in cell lines decreases CREB protein level and induces a lowered clonogenic potential in vitro. It decreases the ability of HL60 to invade the extramedullary sites and to promote bone marrow angiogenesis in nonobese diabetic-severe combined immunodeficient mice, demonstrating its potential effects on tumor progression. ICER represses the majority of 96 target genes upregulated by CREB. It binds CRE promoters and controls gene expression restoring the normal regulation of major cellular pathways. ICER is subjected to degradation through a constitutively active form of the extracellular signal-regulated protein kinase, which drives it to the proteasome. We propose that ICER is downregulated in HL60 to preserve CREB overexpression, which disrupts normal myelopoiesis and promotes blast proliferation. These findings define the function of ICER as a tumor suppressor in leukemia. Unbalanced CREB/ICER expression needs to be considered a pathogenetic feature in leukemogenesis. The molecular characterization of this pathway could be useful for novel therapeutic strategies
miR-34b targets cyclic AMP-responsive element binding protein in acute myeloid leukemia.
No full textThe cyclic AMP-responsive element binding protein (CREB) is documented to be overexpressed in leukemia, but the underlying mechanism remains unknown. Here, microRNAs (miRNA), which act as negative regulators of gene expression principally through translational repression, are investigated for the mediation of high CREB protein levels. A series of miRNAs that target CREB were identified. Real-time quantitative PCR revealed that miR-34b was expressed significantly less in myeloid cell lines, previously known for high CREB protein levels. Exogenous miR-34b expression was induced, and results revealed a direct interaction with the CREB 3'-untranslated region, with the consequent reduction of the CREB protein levels in vitro. miR-34b restored expression caused cell cycle abnormalities, reduced anchorage-independent growth, and altered CREB target gene expression, suggesting its suppressor potential. Using reverse-phase protein array, CREB target proteins (BCL-2, cyclin A1, cyclin B1, cyclin D, nuclear factor-kappaB, Janus-activated kinase 1, and signal transducer and activator of transcription 3), as well as many downstream protein kinases and cell survival signaling pathways (AKT/mammalian target of rapamycin and extracellular signal-regulated kinase) usually elicited by CREB, were observed to have decreased. The miR-34b/miR-34c promoter was shown to be methylated in the leukemia cell lines used. This epigenetic regulation should control the observed miR-34b expression levels to maintain the CREB protein overexpressed. In addition, the inverse correlation between miR-34b and CREB expression was found in a cohort of 78 pediatric patients at diagnosis of acute myeloid leukemia, supporting this relationship in vivo. Our results identify a direct miR-34b target gene, provide a possible mechanism for CREB overexpression, and provide new information about myeloid transformation and therapeutic strategies
Mir-34b promoter methylation plays a role in myeloid transformation by controlling CREB expression.
No full textBAG1: the guardian of anti-apoptotic proteins in acute myeloid leukemia.
Full text linkBCL2 associated Athano-Gene 1 (BAG1) is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance
CREB e ICER: Nuovi regolatori dell'espressione genica dipendente dall'AMP ciclico nella leucemia acuta pediatrica.
No full textMiR 34b Promoter Methylation and Regulation of CREB Expression In Myeloid Transformation
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