Inhibition of melanogenesis in response to oxidative stress: transient downregulation of melanocyte differentiation markers and possible involvement of microphthalmia transcription factor

2001 ◽  
Vol 114 (12) ◽  
pp. 2335-2344 ◽  
Author(s):  
Celia Jiménez-Cervantes ◽  
María Martínez-Esparza ◽  
Cristina Pérez ◽  
Nicole Daum ◽  
Francisco Solano ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Enzyme Activities ◽  
Mrna Levels ◽  
Melanin Synthesis ◽  
Differentiation Markers ◽  
H2o2 Treatment ◽  
Dopachrome Tautomerase ◽  
Protein Levels ◽  
Microphthalmia Transcription Factor

H2O2 and other reactive oxygen species are key regulators of many intracellular pathways. Within mammalian skin, H2O2 is formed as a byproduct of melanin synthesis, and following u.v. irradiation. We therefore analyzed its effects on melanin synthesis. The activity of the rate-limiting melanogenic enzyme, tyrosinase, decreased in H2O2-treated mouse and human melanoma cells. This inhibition was concentration- and time-dependent in the B16 melanoma model. Maximal inhibition (50-75%) occurred 8-16 hours after a 20 minute exposure to 0.5 mM H2O2. B16 cells withstand this treatment adequately, as shown by a small effect on glutathione levels and a rapid recovery of basal lipid peroxidation levels. Enzyme activities also recovered, beginning to increase 16-20 hours after the treatment. Inhibition of enzyme activities reflected decreased protein levels. mRNAs for tyrosinase, tyrosinase-related protein 1, dopachrome tautomerase, silver protein and melanocortin 1 receptor also decreased after H2O2 treatment, and recovered at different rates. Downregulation of melanocyte differentiation markers mRNAs was preceded by a decrease in microphthalmia transcription factor (Mitf) gene expression, which was quantitatively similar to the decrease achieved using 12-O-tetradecanoylphorbol-13-acetate. Recovery of basal Mitf mRNA levels was also observed clearly before that of tyrosinase. Therefore, oxidative stress may lead to hypopigmentation by mechanisms that include a microphthalmia-dependent downregulation of the melanogenic enzymes.

scholarly journals Novel Interplay between p53 and HO-1 in Embryonic Stem Cells

Cells ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 35
Author(s):  
Ayelén Toro ◽  
Nicolás Anselmino ◽  
Claudia Solari ◽  
Marcos Francia ◽  
Camila Oses ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Cellular Response ◽  
Es Cells ◽  
Embryonic Stem ◽  
Protective Role ◽  
Heme Oxygenase 1 ◽  
Mrna Levels ◽  
H2o2 Treatment ◽  
Protein Levels

Stem cells genome safeguarding requires strict oxidative stress control. Heme oxygenase-1 (HO-1) and p53 are relevant components of the cellular defense system. p53 controls cellular response to multiple types of harmful stimulus, including oxidative stress. Otherwise, besides having a protective role, HO-1 is also involved in embryo development and in embryonic stem (ES) cells differentiation. Although both proteins have been extensively studied, little is known about their relationship in stem cells. The aim of this work is to explore HO-1-p53 interplay in ES cells. We studied HO-1 expression in p53 knockout (KO) ES cells and we found that they have higher HO-1 protein levels but similar HO-1 mRNA levels than the wild type (WT) ES cell line. Furthermore, cycloheximide treatment increased HO-1 abundance in p53 KO cells suggesting that p53 modulates HO-1 protein stability. Notably, H2O2 treatment did not induce HO-1 expression in p53 KO ES cells. Finally, SOD2 protein levels are also increased while Sod2 transcripts are not in KO cells, further suggesting that the p53 null phenotype is associated with a reinforcement of the antioxidant machinery. Our results demonstrate the existence of a connection between p53 and HO-1 in ES cells, highlighting the relationship between these stress defense pathways.

scholarly journals GIF-2209, an Oxindole Derivative, Accelerates Melanogenesis and Melanosome Secretion via the Modification of Lysosomes in B16F10 Mouse Melanoma Cells

Molecules ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 177
Author(s):  
Miyu Watanabe ◽  
Kyoka Kawaguchi ◽  
Yusuke Nakamura ◽  
Kyoji Furuta ◽  
Hiroshi Takemori
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Mrna Levels ◽  
Related Protein ◽  
Mouse Melanoma ◽  
Protein Levels ◽  
Independent Mechanism ◽  
Microphthalmia Transcription Factor ◽  
Tyr Gene ◽  
Tyrosinase Related Protein

Melanogenesis and melanosome secretion are regulated by several mechanisms. In this study, we found that the oxindole derivative GIF-2209 accelerated melanogenesis associated with the discrimination in the expression and intracellular distributions of two melanogenic enzymes, tyrosinase (TYR) and tyrosinase-related protein-1 (TYRP-1). GIF-2209 upregulated the expression of TYR via a microphthalmia transcription factor (MITF)-independent mechanism, leading to high expression of protein. In contrast, GIF-2209 did not alter the mRNA levels of TYRP-1 and suppressed its protein levels. GIF-2209 induced the dissociation of TYR from TYRP-1 but did not alter the association between TYR and CD63, a melanosome and lysosome marker. The protein levels of CD63 were also upregulated by GIF-2209. GIF-2209 induced lysosome expansion and redistribution in all areas of the cytosol, accompanied by autophagy acceleration (upregulation of LC3BII protein levels and downregulation of p62 protein levels). In addition, GIF-2209 stimulated the secretion of melanosomes containing high levels of TYR, TYRP-1, and CD63 proteins. The GIF-2209 mediated melanosome secretion was sensitive to the lysosome inhibitor chloroquine. These results suggest that GIF-2209 may activate lysosomal functions with TYR gene expression, while it accelerates melanosome secretion, which finally leads to the depletion of intracellular melanogenic enzyme, especially TYRP-1 protein.

scholarly journals Relationships of Telomere Homeostasis with Oxidative Stress and Cardiac Dysfunction in Human Ischaemic Hearts

Antioxidants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1750
Author(s):  
Estefanía Tarazón ◽  
Lorena Pérez-Carrillo ◽  
Isaac Giménez-Escamilla ◽  
Pablo Ramos-Castellanos ◽  
Luis Martínez-Dolz ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Repair ◽  
Transcription Factor ◽  
Cardiac Dysfunction ◽  
Mrna Level ◽  
Left Ventricular ◽  
Mrna Levels ◽  
Telomeric Dna ◽  
Protein Levels ◽  
Telomere Homeostasis

Although the roles of telomeres and oxidative stress in ischaemic cardiomyopathy (ICM) are known, mechanisms of telomere homeostasis and their relationship with oxidative stress are incompletely understood. We performed two RNA-seq analyses (mRNA n = 23; ncRNA n = 30) and protein validation on left ventricles of explanted hearts from ICM and control subjects. We observed dysregulation of the shelterin and cohesin complexes, which was related to an increase in the response to cellular oxidative stress. Moreover, we found alterations at mRNA level in the mechanisms of telomeric DNA repair. Specifically, increased RAD51D mRNA levels were correlated with left ventricular diameters. RAD51D protein levels were unaltered, however, and were inversely corelated with the miR-103a-3p upregulation. We also observed the overexpression of lncRNAs (TERRA and GUARDIN) involved in telomere protection in response to stress and alterations in their regulatory molecules. Expression of the TERRA transcription factor ATF7 was correlated with superoxide dismutase 1 expression and left ventricular diameters. The levels of GUARDIN and its transcription factor FOSL2 were correlated with those of catalase. Therefore, we showed specific alterations in the mechanisms of telomeric DNA repair and protection, and these alterations are related to an increase in the response mechanisms to oxidative stress and cardiac dysfunction in ICM.

scholarly journals The Role of the FOXO1/β2-AR/p-NF-κB p65 Pathway in the Development of Endometrial Stromal Cells in Pregnant Mice under Restraint Stress

2021 ◽  
Vol 22 (3) ◽  
pp. 1478
Author(s):  
Jiayin Lu ◽  
Yaoxing Chen ◽  
Zixu Wang ◽  
Jing Cao ◽  
Yulan Dong
Keyword(s):  
Oxidative Stress ◽  
Stromal Cells ◽  
Restraint Stress ◽  
Target Genes ◽  
Mrna Levels ◽  
Endometrial Stromal Cells ◽  
Protein Levels ◽  
Pregnant Mice

Restraint stress causes various maternal diseases during pregnancy. β2-Adrenergic receptor (β2-AR) and Forkhead transcription factor class O 1 (FOXO1) are critical factors not only in stress, but also in reproduction. However, the role of FOXO1 in restraint stress, causing changes in the β2-AR pathway in pregnant mice, has been unclear. The aim of this research was to investigate the β2-AR pathway of restraint stress and its impact on the oxidative stress of the maternal uterus. In the study, maternal mice were treated with restraint stress by being restrained in a transparent and ventilated device before sacrifice on Pregnancy Day 5 (P5), Pregnancy Day 10 (P10), Pregnancy Day 15 (P15), and Pregnancy Day 20 (P20) as well as on Non-Pregnancy Day 5 (NP5). Restraint stress augmented blood corticosterone (CORT), norepinephrine (NE), and blood glucose levels, while oestradiol (E2) levels decreased. Moreover, restraint stress increased the mRNA levels of the FOXO family, β2-AR, and even the protein levels of FOXO1 and β2-AR in the uterus and ovaries. Furthermore, restraint stress increased uterine oxidative stress level. In vitro, the protein levels of FOXO1 were also obviously increased when β2-AR was activated in endometrial stromal cells (ESCs). In addition, phosphorylated-nuclear factor kappa-B p65 (p-NF-κB p65) and its target genes decreased significantly when FOXO1 was inhibited. Overall, it can be said that the β2-AR/FOXO1/p-NF-κB p65 pathway was activated when pregnant mice were under restraint stress. This study provides a scientific basis for the origin of psychological stress in pregnant women.

PERK mediates oxidative stress and adipogenesis in Graves’ orbitopathy pathogenesis

2021 ◽  
Author(s):  
JaeSang Ko ◽  
Ji-Young Kim ◽  
Min Kyung Chae ◽  
Eun Jig Lee ◽  
Jin Sook Yoon
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Er Stress ◽  
Adipogenic Differentiation ◽  
Ros Generation ◽  
Mrna Levels ◽  
Graves Orbitopathy ◽  
Orbital Fibroblasts

We examined endoplasmic reticulum (ER) stress-related gene expression in orbital tissues from patients with Graves’ orbitopathy (GO) and the effects of silencing protein kinase RNA-like endoplasmic reticulum kinase (PERK) in primary orbital fibroblast cultures to demonstrate the therapeutic potential of PERK-modulating agents in GO management. The expression of ER stress related genes in orbital tissue harvested from individuals with or without GO was studied using real-time polymerase chain reaction. The role of PERK in GO pathogenesis was examined through small-interfering RNA (siRNA)-mediated silencing in cultured primary orbital fibroblasts. Intracellular reactive oxygen species (ROS) levels induced in response to cigarette smoke extract (CSE) or hydrogen peroxide were measured using 5-(and 6)-carboxy-20,70-dichlorodihydrofluorescein diacetate staining and flow cytometry. Cells were stained with Oil Red O, and adipogenesis-related transcription factor expression was evaluated through western blotting after adipogenic differentiation. PERK, activating transcription factor 4 (ATF4), and CCAAT-enhancer-binding protein (C/EBP)-homologous protein(CHOP)mRNA levels were significantly higher in GO orbital tissues than in non-GO orbital tissues. PERK silencing inhibited CSE- or hydrogen peroxide-induced ROS generation. After adipogenic differentiation, GO orbital fibroblasts revealed decreased lipid droplets and downregulation of C/EBPα, C/EBPβ, and peroxisome proliferator-activator gamma (PPARγ) in PERK siRNA-transfected cells. The orbital tissues of patients with GO were exposed to chronic ER stress and subsequently exhibited enhanced unfolded protein response (especially through the PERK pathway). PERK silencing reduced oxidative stress and adipogenesis in GO orbital fibroblasts in vitro. Our results imply that PERK-modulating agents can potentially be used to manage GO.

scholarly journals Induction of Oxidative Stress and Apoptosis in the Injured Brain: Potential Relevance to Brain Regeneration in Zebrafish.

2021 ◽  
Author(s):  
Surendra Kumar Anand ◽  
Manas Ranjan Sahu ◽  
Amal Chandra Mondal
Keyword(s):  
Oxidative Stress ◽  
Mrna Levels ◽  
Adult Zebrafish ◽  
Zebrafish Model ◽  
Stab Injury ◽  
Zebrafish Brain ◽  
Protein Levels ◽  
Injured Brain ◽  
Brain Regeneration ◽  
The Impact

Abstract In the recent years, zebrafish, owing to its tremendous adult neurogenic capacity, has emerged as a useful vertebrate model to study brain regeneration. Recent findings suggest a significant role of the BDNF/TrkB signaling as a mediator of brain regeneration following a stab injury in the adult zebrafish brain. Since BDNF has been implicated in a plethora of physiological processes, we hypothesized that these processes are affected in the injured zebrafish brain. In this small study, we examined the indicators of oxidative stress and of apoptosis using biochemical assays, RT-PCR and IHC to reflect upon the impact of stab injury on oxidative stress levels and apoptosis in the injured adult zebafish brain. Our results indicate induction of oxidative stress in the injured adult zebrafish brain. Also, apoptosis was induced in the injured brain as indicated by increased protein levels of cleaved caspase3 as well as enhanced mRNA levels of both pro-apoptotic and anti-apoptotic genes. This knowledge contributes to the overall understanding of adult neurogenesis in the zebrafish model and raises new questions pertaining to the compensatory physiological mechanisms in response to traumatic brain injury in the adult zebrafish brain.

scholarly journals Recuperating Lung Decoction Attenuates the Oxidative Stress State of Chronic Obstructive Pulmonary Disease by Inhibiting the MAPK/AP-1 Signal Pathway and Regulating γ-GCS

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Chunlei Li ◽  
Qi Shi ◽  
Yue Yan ◽  
Yanhua Kong ◽  
YanYan Meng ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mapk Pathway ◽  
Signal Pathway ◽  
P38 Map Kinase ◽  
Chronic Obstructive ◽  
Mrna Levels ◽  
Obstructive Pulmonary Disease ◽  
Protein Levels ◽  
Extracellular Signal ◽  
Cysteine Synthetase

Purpose/Objective. To evaluate the effects of Recuperating Lung Decoction (RLD) on the indices of oxidative stress in a rat model of COPD and detect the indices of the MAPK/AP-1/γ-GCS signal pathway for a further survey of the possible targeting site of RLD. Methods/Materials. The rats of COPD were treated with RLD. The protein levels of glutathione (GSH), oxidized glutathione (GSSG), 8-hydroxy-2-deoxyguanosine (8-OHdG), and 4-hydroxynonenal (4-HNE) were measured. In addition, the levels of key signaling molecules (extracellular signal-regulated kinases [ERK], the c-jun N-terminal kinase [JNKs signal pathway], and p38 MAP kinase [p38MAPK], AP-1 proteins [C-fos, C-jun], and γ-glutamyl-cysteine synthetase [γ-GCS-h]) of the MAPK/AP-1/γ-GCS-h signal pathway were assessed. Results. After treatment, the protein level of GSH and the ratio of GSH/GSSG were increased and the amounts of 8-OHdG and 4-HNE were decreased significantly in lung tissues when compared with the nontreated COPD group. Further results showed that the RLD could effectively inhibit the MAPK pathway by inactivation of p38MAPK and ERK and could also downregulate the AP-1 and the γ-GCS-h genes expressions in both protein and mRNA levels. Conclusion. RLD might improve the state of oxidative stress by downregulation of the expression of γ-GCS-h gene by inhibition of the MAPK/AP-1 pathway, thereafter enhancing the ability of antioxidation in COPD.

scholarly journals Evolutionarily conserved transcription factors drive the oxidative stress response in Drosophila

2020 ◽  
Vol 223 (14) ◽  
pp. jeb221622
Author(s):  
Sarah M. Ryan ◽  
Kaitie Wildman ◽  
Briseida Oceguera-Perez ◽  
Scott Barbee ◽  
Nathan T. Mortimer ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Stress Responses ◽  
Binding Sites ◽  
Regulatory Elements ◽  
Genomic Locus ◽  
Biologically Relevant ◽  
Protein Levels ◽  
Putative Transcription Factor

ABSTRACTAs organisms are constantly exposed to the damaging effects of oxidative stress through both environmental exposure and internal metabolic processes, they have evolved a variety of mechanisms to cope with this stress. One such mechanism is the highly conserved p38 MAPK (p38K) pathway, which is known to be post-translationally activated in response to oxidative stress, resulting in the activation of downstream antioxidant targets. However, little is known about the role of p38K transcriptional regulation in response to oxidative stress. Therefore, we analyzed the p38K gene family across the genus Drosophila to identify conserved regulatory elements. We found that oxidative stress exposure results in increased p38K protein levels in multiple Drosophila species and is associated with increased oxidative stress resistance. We also found that the p38Kb genomic locus includes conserved AP-1 and lola-PT transcription factor consensus binding sites. Accordingly, over-expression of these transcription factors in D. melanogaster is sufficient to induce transcription of p38Kb and enhances resistance to oxidative stress. We further found that the presence of a putative lola-PT binding site in the p38Kb locus of a given species is predictive of the species' survival in response to oxidative stress. Through our comparative genomics approach, we have identified biologically relevant putative transcription factor binding sites that regulate the expression of p38Kb and are associated with resistance to oxidative stress. These findings reveal a novel mode of regulation for p38K genes and suggest that transcription may play as important a role in p38K-mediated stress responses as post-translational modifications.

TPO-Induced miR-150 Down-Regulates c-Myb in Primary Megakaryocytes and a Megakaryocytic Cell Line.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1238-1238 ◽  
Author(s):  
Charlene F. Barroga ◽  
Hang Pham ◽  
Kenneth Kaushansky
Keyword(s):  
Transcription Factor ◽  
Western Blotting ◽  
Luciferase Activity ◽  
Mrna Translation ◽  
Negative Control ◽  
Luciferase Reporter ◽  
Translation Efficiency ◽  
Mrna Levels ◽  
Protein Levels ◽  
Micro Rnas

Abstract Mice harboring c-Myb hypomorphic mutations display enhanced thrombopoiesis because of increased numbers of megakaryocytic progenitors (CFU-MK) and mature megakaryocytes (MK). Thrombopoietin (Tpo), the primary regulator of megakaryopoiesis, induces these same effects, which lead us to hypothesize that Tpo might act, at least in part, through modulation of c-Myb expression. We found using quantitative (Q)-PCR that c-Myb mRNA levels were 13-fold reduced during Tpo-induced MK maturation. Micro RNAs (miRs) are ∼22 nucleotide species that down-regulate gene expression by binding to the 3′ untranslated region (UTR) of specific mRNAs, enhancing mRNA degradation, or by reducing mRNA translation efficiency. We noted that the 3′UTR of c-Myb contains a number of miR target sites, including four that bind miR150; using a specific Q-PCR assay we also found that Tpo increased mir-150 expression to 160% of baseline at 24 hr and 250% at 48 hr in UT7/TPO cells (n=2 experiments). To test if miR150 affects c-Myb expression, we introduced the 3′UTR of c-Myb into a luciferase reporter gene (pCMV-luc-3′UTRcMyb), in which CMV promoter-driven luciferase activity would reflect the stability of the 3′UTR of c-Myb, and allow us to test the effects of miR150 on c-Myb expression in transduced cells; Q-PCR and western blotting were used to simultaneously assess endogenous c-Myb mRNA and protein levels in the cells treated with miR-150 and anti-miR-150, and their respective controls (Ambion, ABI). Co-transfection of UT7/TPO cells with pCMV-luc-3′UTRcMyb and miR-150 significantly down-regulated luciferase activity to 40% of baseline 24 hr following transfection (p = 0.035; n=2 experiments) compared to a miR negative control. Luciferase activity in cells transfected with a control luc plasmid lacking the 3′UTR of c-Myb was not modulated by introduction of miR-150. Q-PCR analysis revealed that endogenous c-Myb mRNA was significantly down-regulated to 60% of baseline upon transfection of miR-150 compared to the negative control (p = 0.043), while the essential megakaryocytic transcription factor, AML1/RUNX1, remained unaltered. Western blotting of these cell lysates revealed that c-Myb protein expression was down-regulated to 30% of baseline (n=3 experiments) following transduction with miR150 but not with the miR negative control. Converse experiments utilizing anti-miRs, which inhibit expression of endogenous miRs, revealed that anti-miR150 significantly upregulated luciferase activity to 180% of baseline compared to an anti-miR-negative control (p=0.003; n=2 experiments). These findings establish that miR-150 down-modulates c-Myb mRNA, and to a greater extent protein levels, suggesting effects on both mRNA stability and protein translation efficiency. And since Tpo affects miR-150 expression, our results also suggest that in addition to direct effects on the survival and growth of MK progenitor cells, mediated by the JAK/STAT, PI3K/Akt and MAPK pathways, Tpo down-modulates c-Myb expression during megakaryopoiesis through the induction of miR150. We are currently ascertaining the in vivo role of miR-150 in Tpo-induced megakaryopoiesis, but these studies already establish that hematopoietic growth factors such as Tpo can influence transcription factor expression through modulation of microRNA species.

Requirement of the E2F3 Transcription Factor for BCR/ABL Leukemogenesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 33-33
Author(s):  
Anna M. Eiring ◽  
Paolo Neviani ◽  
Ramasamy Santhanam ◽  
Joshua J. Oaks ◽  
Ji Suk Chang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Blast Crisis ◽  
Scid Mice ◽  
Myelogenous Leukemia ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Hnrnp A1 ◽  
Protein Levels ◽  
Abl Kinase

Abstract Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are altered at transcriptional or post-translational levels by the increased constitutive kinase activity of the BCR/ABL oncoprotein, resulting in enhanced resistance to apoptotic stimuli, growth advantage and differentiation arrest of CD34+ CML blast crisis (CML-BC) progenitors. In the current study, we identified by RIP (RNA immunoprecipitation)-mediated microarray analysis that mRNA encoding the E2F3 transcription factor associates to the BCR/ABL-regulated RBP hnRNP A1. Moreover, RNA electrophoretic mobility shift and UV-crosslinking assays revealed that hnRNP A1 interacts with E2F3 mRNA through a binding site located in the 3’UTR of both human and mouse E2F3 mRNA. Accordingly, E2F3 protein levels were upregulated in BCR/ABL-transformed myeloid precursor cell lines compared to parental cells in a BCR/ABL-kinase- and hnRNP A1 shuttling-dependent manner. In fact, treatment of BCR/ABL-expressing myeloid precursors with the kinase inhibitor Imatinib (2mM, 24 hr) or introduction of a dominant-negative shuttling-deficient hnRNP A1 protein (NLS-A1) markedly reduced E2F3 protein and mRNA levels. Similarly, upregulation of BCR/ABL expression/activity in the doxycycline inducible TonB2.10 cell line resulted in increased E2F3 protein expression. BCR/ABL kinase-dependent induction of E2F3 protein levels was also detected in CML-BCCD34+ compared to CML-CPCD34+ progenitors from paired patient samples and to normal CD34+ bone marrow samples. Importantly, the in vitro clonogenic potential of primary mouse BCR/ABL+ lineage negative (Lin−) progenitors was markedly impaired in BCR/ABL+ E2F3−/− compared to BCR/ABL-transduced E2F3+/+ myeloid progenitors and upon shRNA-mediated downregulation of E2F3 expression (90% inhibition, P<0.001). Furthermore, subcutaneous injection of shE2F3-expressing BCR/ABL+ cells into SCID mice markedly impaired in vivo tumorigenesis (>80% reduction in tumor burden, P<0.01). Accordingly, BCR/ABL leukemogenesis was strongly inhibited in SCID mice intravenously injected with E2F3 shRNA-expressing 32D-BCR/ABL cells and in mice transplanted with BCR/ABL-transduced Lin− bone marrow cells from E2F3−/− mice. Specifically, we demonstrate that reduced or absent levels of E2F3 resulted in dramatically decreased numbers of circulating BCR/ABL+ cells as determined by nested RT-PCR at 4 weeks post-injection (P=0.0001), normal splenic architecture and bone marrow cellularity and the absence of infiltrating myeloid blasts into non-hematopoietic compartments (i.e. liver). By contrast, SCID mice transplanted with vector-transduced 32D-BCR/ABL cells or BCR/ABL+ E2F3+/+ Lin− BM progenitors showed signs of an overt acute leukemia-like process with blast infiltration of hematopoietic and non-hematopoietic organs. Altogether, these data outline the importance of E2F3 expression for BCR/ABL leukemogenesis and characterize a new potential therapeutic target for the treatment of patients with advanced phase CML.

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