scholarly journals Forkhead Transcription Factor FKHR-L1 Modulates Cytokine-Dependent Transcriptional Regulation of p27KIP1

2000 ◽  
Vol 20 (24) ◽  
pp. 9138-9148 ◽  
Author(s):  
Pascale F. Dijkers ◽  
Rene H. Medema ◽  
Cornelieke Pals ◽  
Lolita Banerji ◽  
N. Shaun B. Thomas ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Cell Survival ◽  
Kinase Inhibitor ◽  
Ectopic Expression ◽  
Dependent Manner ◽  
Forkhead Transcription Factor ◽  
Protein Levels ◽  
Macrophage Colony ◽  
Null Mutant Mice

ABSTRACT Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27KIP1 through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27KIP1 promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27KIP1 appears to be critical in the regulation of cell survival since mere ectopic expression of p27KIP1 was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27KIP1 null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27KIP1transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.

scholarly journals Mitochondrial Deoxyguanosine Kinase Regulates NAD+ Biogenesis Independent of Mitochondria Complex I Activity

2020 ◽  
Vol 10 ◽  
Author(s):  
Lei Sang ◽  
Ying-Jie He ◽  
Jiaxin Kang ◽  
Hongyi Ye ◽  
Weiyu Bai ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cancer Progression ◽  
Complex I ◽  
Ectopic Expression ◽  
Dependent Manner ◽  
Potential Therapeutic Target ◽  
Protein Levels ◽  
Deoxyguanosine Kinase ◽  
Complex I Activity ◽  
Mitochondria Complex I

Overexpression of DGUOK promotes mitochondria oxidative phosphorylation and lung adenocarcinoma progression. However, the role and mechanism of DGUOK in regulation of mitochondria function and lung cancer progression still poorly understood. Here we demonstrated that DGUOK regulated NAD+ biogenesis. Depletion of the DGUOK significantly decreased NAD+ level. Furthermore, knockout of the DGUOK considerably reduced expression of the NMNAT2, a key molecule controlling NAD+ synthesis, at both mRNA and protein levels. Ectopic expression of the NMNAT2 abrogated the effect of knockdown of DGUOK on NAD+. Notably, this regulation is independent of DGUOK -mediated mitochondria complex I activity. We also showed that NMNAT2 was highly expressed in lung adenocarcinoma and negatively correlated with the patient overall survival. Our study suggested that DGUOK regulates NAD+ in a NMNAT2 dependent manner and DGUOK-NMNAT2-NAD+ axis could be a potential therapeutic target in lung adenocarcinoma.

IRF6 and TAK1 coordinately promote the activation of HIPK2 to stimulate apoptosis during palate fusion

2019 ◽  
Vol 12 (593) ◽  
pp. eaav7666 ◽  
Author(s):  
Chen-Yeh Ke ◽  
Hua-Hsuan Mei ◽  
Fen-Hwa Wong ◽  
Lun-Jou Lo
Keyword(s):  
Transcription Factor ◽  
Cell Apoptosis ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Human Cancer ◽  
Ectopic Expression ◽  
Interacting Protein ◽  
Human Cancer Cells

Cleft palate is a common craniofacial defect caused by a failure in palate fusion. The palatal shelves migrate toward one another and meet at the embryonic midline, creating a seam. Transforming growth factor–β3 (TGF-β3)–induced apoptosis of the medial edge epithelium (MEE), the cells located along the seam, is required for completion of palate fusion. The transcription factor interferon regulatory factor 6 (IRF6) promotes TGF-β3–induced MEE cell apoptosis by stimulating the degradation of the transcription factor ΔNp63 and promoting the expression of the gene encoding the cyclin-dependent kinase inhibitor p21. Because homeodomain-interacting protein kinase 2 (HIPK2) functions downstream of IRF6 in human cancer cells and is required for ΔNp63 protein degradation in keratinocytes, we investigated whether HIPK2 played a role in IRF6-induced ΔNp63 degradation in palate fusion. HIPK2 was present in the MEE cells of mouse palatal shelves during seam formation in vivo, and ectopic expression of IRF6 in palatal shelves cultured ex vivo stimulated the expression of Hipk2 and the accumulation of phosphorylated HIPK2. Knockdown and ectopic expression experiments in organ culture demonstrated that p21 was required for HIPK2- and IRF6-dependent activation of caspase 3, MEE apoptosis, and palate fusion. Contact between palatal shelves enhanced the phosphorylation of TGF-β–activated kinase 1 (TAK1), which promoted the phosphorylation of HIPK2 and palate fusion. Our findings demonstrate that HIPK2 promotes seam cell apoptosis and palate fusion downstream of IRF6 and that IRF6 and TAK1 appear to coordinately enhance the abundance and activation of HIPK2 during palate fusion.

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.

scholarly journals Foxl2, a Forkhead Transcription Factor, Modulates Nonclassical Activity of the Estrogen Receptor-α

Endocrinology ◽  
2009 ◽  
Vol 150 (11) ◽  
pp. 5085-5093 ◽  
Author(s):  
So-Youn Kim ◽  
Jeffrey Weiss ◽  
Minghan Tong ◽  
Monica M. Laronda ◽  
Eun-Jig Lee ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Transcription Factor ◽  
Direct Action ◽  
Ovarian Follicle ◽  
Estrogen Receptor Α ◽  
Ovary Development ◽  
Activator Protein 1 ◽  
Forkhead Transcription Factor ◽  
Protein Levels ◽  
And Function

Foxl2 is a forkhead transcription factor required for ovary development and ovarian follicle maturation. In this report, we identified and characterized a functional relationship between Foxl2 expression and estrogen receptor (ER)-α signaling. We show that Foxl2 has no effect on classical ERα-mediated transcription, which occurs through canonical estrogen response elements. However, Foxl2 suppresses ERα signaling through nonclassical tethered transcriptional pathways. Specifically, the selective ER modulator tamoxifen stimulates activator protein-1 (AP1)-dependent transcription via the ERα, and this enhancement is blocked by Foxl2. Two lines of evidence suggest that Foxl2 suppression is mediated by physical interactions with ERα rather than direct action at AP1 binding sites. First, ERα is coimmunoprecipitated with Foxl2. Second, activation of a upstream activating sequence (UAS) reporter by Gal4-cJun in the presence of ERα and tamoxifen was blocked by Foxl2, demonstrating suppression in the absence of an AP1 site. Cyclooxygenase-2 (COX2), which is required for ovulation, was identified through expression profiling as a candidate physiological target for nonclassical ERα signaling and thus modulation by ERα/Foxl2 interactions. This possibility was confirmed by two sets of experiments. COX2 protein levels were induced by ERα in the presence of tamoxifen, and protein expression was suppressed by Foxl2. In addition, ERα stimulation of the COX2 promoter was repressed by Foxl2. We conclude that ERα and Foxl2 interact and that Foxl2 selectively suppresses ERα-mediated transcription of AP1-regulated genes. These data provide a potential point of convergence for ERα and Foxl2 to regulate ovarian development and function.

scholarly journals Gain-of-function genetic screen of the kinome reveals BRSK2 as an inhibitor of the NRF2 transcription factor

2019 ◽  
Author(s):  
Tigist Y Tamir ◽  
Brittany M Bowman ◽  
Megan J Agajanian ◽  
Dennis Goldfarb ◽  
Travis P Schrank ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Synthesis ◽  
Human Cancer ◽  
Genetic Screen ◽  
Mtor Signaling ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Gain Of Function ◽  
Protein Levels ◽  
Nrf2 Protein

AbstractNFE2L2/NRF2 is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, and conversely NRF2 protein levels as well as activity is diminished with age and in neurodegenerative disorders. Though NRF2 activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here we used a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the understudied protein kinase Brain Specific Kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives AMPK activation and suppresses mTOR signaling. As a result, BRSK2 kinase activation suppressed ribosome-RNA complexes, global protein synthesis, and NRF2 protein levels. Collectively, our data establish the catalytically active BRSK2 kinase as a negative regulator of NRF2 via the AMPK/mTOR signaling. This signaling axis may prove useful for therapeutically targeting NRF2 in human diseases.Summary StatementBRSK2 suppresses NRF2 signaling by inhibiting protein synthesis through mTOR downregulation.

scholarly journals Gain-of-function genetic screen of the kinome reveals BRSK2 as an inhibitor of the NRF2 transcription factor

2020 ◽  
Vol 133 (14) ◽  
pp. jcs241356 ◽  
Author(s):  
Tigist Y. Tamir ◽  
Brittany M. Bowman ◽  
Megan J. Agajanian ◽  
Dennis Goldfarb ◽  
Travis P. Schrank ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Human Cancer ◽  
Antioxidant Response ◽  
Genetic Screen ◽  
Related Factor ◽  
Dependent Manner ◽  
Gain Of Function ◽  
Protein Levels ◽  
Nrf2 Protein

ABSTRACTNuclear factor erythroid 2-related factor 2 (NFE2L2, also known as NRF2) is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, but conversely NRF2 activity diminishes with age and in neurodegenerative and metabolic disorders. Although NRF2-activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here, we describe use of a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the under-studied protein kinase brain-specific kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives 5′-AMP-activated protein kinase α2 (AMPK) signaling and suppresses the mTOR pathway. As a result, BRSK2 kinase activation suppresses ribosome-RNA complexes, global protein synthesis and NRF2 protein levels. Collectively, our data illuminate the BRSK2 and BRSK1 kinases, in part by functionally connecting them to NRF2 signaling and mTOR. This signaling axis might prove useful for therapeutically targeting NRF2 in human disease.This article has an associated First Person interview with the first author of the paper.

Arylsulfatase A, a genetic modifier of Parkinson’s disease, is an α-synuclein chaperone

Brain ◽  
2019 ◽  
Vol 142 (9) ◽  
pp. 2845-2859 ◽  
Author(s):  
Jun Sung Lee ◽  
Kazuaki Kanai ◽  
Mari Suzuki ◽  
Woojin S Kim ◽  
Han Soo Yoo ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Genetic Modifier ◽  
Ectopic Expression ◽  
Physical Interaction ◽  
Dependent Manner ◽  
Arylsulfatase A ◽  
Lysosomal Storage ◽  
Protein Levels

AbstractMutations in lysosomal genes increase the risk of neurodegenerative diseases, as is the case for Parkinson’s disease. Here, we found that pathogenic and protective mutations in arylsulfatase A (ARSA), a gene responsible for metachromatic leukodystrophy, a lysosomal storage disorder, are linked to Parkinson’s disease. Plasma ARSA protein levels were changed in Parkinson’s disease patients. ARSA deficiency caused increases in α-synuclein aggregation and secretion, and increases in α-synuclein propagation in cells and nematodes. Despite being a lysosomal protein, ARSA directly interacts with α-synuclein in the cytosol. The interaction was more extensive with protective ARSA variant and less with pathogenic ARSA variant than wild-type. ARSA inhibited the in vitro fibrillation of α-synuclein in a dose-dependent manner. Ectopic expression of ARSA reversed the α-synuclein phenotypes in both cell and fly models of synucleinopathy, the effects correlating with the extent of the physical interaction between these molecules. Collectively, these results suggest that ARSA is a genetic modifier of Parkinson’s disease pathogenesis, acting as a molecular chaperone for α-synuclein.

The patched signaling pathway mediates repression of gooseberry allowing neuroblast specification by wingless during Drosophila neurogenesis

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2921-2932 ◽  
Author(s):  
K.M. Bhat
Keyword(s):  
Nervous System ◽  
Transcription Factor ◽  
Signaling Pathway ◽  
Transmembrane Protein ◽  
Ectopic Expression ◽  
Negative Regulation ◽  
Dependent Manner ◽  
Neuronal Precursor ◽  
System A ◽  
Developing Nervous System

The Drosophila signaling molecule Wingless (Wg) plays crucial roles in cell-cell communications during development. In the developing nervous system, a previous study has shown that Wg acts non-autonomously to specify the fate of a specific neuronal precursor, NB4-2 (Q. Chu-LaGraff and C. Q. Doe (1993) Science 261, 1594–1597). The lack of autocrine specification of NB4-2 in Wg-expressing cells suggests that the response to Wg is spatially restricted, presumably through the activity of the Wg-receptor. I show that two other proteins, a transcription factor Gooseberry (Gsb) and a transmembrane protein Patched (Ptc), participate in the Wg-mediated specification of NB4-2 by controlling the response to the Wg signal. In gsb mutants, Wg-positive NB5-3 is transformed to NB4-2 in a Wg-dependent manner, suggesting that Gsb normally represses the capacity to respond to the Wg signal. In ptc mutants, Gsb is ectopically expressed in normally Wg-responsive cells, thus preventing the Wg response and consequently the correct specification of NB4-2 does not take place. This conclusion is supported by the observation that NB4-2 can be specified in gsb;ptc double mutants in a Wg-dependent manner. Moreover, ectopic expression of Gsb from the hsp7O-gsb transgene also blocks the response to the Wg signal. I propose that the responsiveness to the Wg signal is controlled by sequential negative regulation, ptc--&gt;gsb--&gt;Wg receptor. The timing of the response to Gsb suggests that the specification of neuroblast identities takes place within the neuroectoderm, prior to neuroblast delamination.

p210BCR-ABL by down Regulating FLI-1 Reprograms Megakaryocytic Progenitors to the Erythroid Lineage.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2275-2275
Author(s):  
Dorothée Buet ◽  
Hana Raslova ◽  
Gaëtan Juban ◽  
Jean-François Geay ◽  
Peggy Jarrier ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Progenitor Cells ◽  
Kinase Inhibitor ◽  
Ectopic Expression ◽  
Erythroid Differentiation ◽  
Culture Conditions ◽  
Abl Tyrosine Kinase ◽  
Human Cd34 ◽  
Key Factor ◽  
Abl Tyrosine Kinase Inhibitor

Abstract Blood cells derive from a common multipotent stem cell that undergoes successive commitment steps with progressive restriction in differentiation ability. A close developmental relationship exists between the erythroid and megakaryocytic (MK) lineages as both lineages derive from a common bipotent progenitor. The nature of the events governing erythroid-specific or MK-specific programming is yet poorly understood. To approach this issue, we used an ectopic expression of the p210 BCR-ABL oncoprotein in adult primary human CD34+ progenitor cells. In culture conditions favoring the development of both erythroid and MK cells, we observed a major expansion of the erythroid lineage occurring at the expense of the MK lineage. The combined use of a p210BCR-ABL tyrosine kinase inhibitor together with single cell cloning experiments on prospectively isolated CD41+CD42− progenitor cells allowed us to identify that a part of MK progenitor cells were reprogrammed by p210BCR-ABL to take an erythroid fate. A similar lineage switching was seen after p210BCR-ABL expression in the human megakaryoblastic MO7e cell line. Microarrays and semi-quantitative RT-PCR studies showed that expression of p210BCR-ABL was associated with a decreased expression of the FLI-1 transcription factor. Moreover, re-expression of FLI-1 in p210BCR-ABL-transduced MO7e cells that had acquired an erythroid phenotype rescued the MK programming. Finally, a partial inhibition of FLI-1 in normal CD41+CD42− progenitor cells with a shRNAi led to an increased erythroid differentiation at the expense of MK differentiation. These results demonstrate that progenitor cells apparently committed to the MK lineage may be re-specified to the erythroid lineage by an ectopic expression of p210BCR-ABL and that the FLI-1 transcription factor is a key factor in the balance between erythroid versus MK commitment.

Inhibitors Of Src Family and AKT Regulate The Activity Of CD20 Promoter

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1838-1838
Author(s):  
Beata Pyrzynska ◽  
Kamil Bojarczuk ◽  
Magdalena Winiarska ◽  
Jacek Bil ◽  
Nina Miazek ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
B Cells ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Akt Kinase ◽  
Cd20 Antigen ◽  
Cd20 Expression ◽  
Anti Cd20 ◽  
Dasatinib Treatment

Abstract Introduction The monoclonal antibodies against CD20 antigen (rituximab and ofatumumab) have been developed and used in a clinic as a therapeutic strategy in B-cell malignancies. These antibodies eliminate B cells by triggering indirect effector mechanisms of the immune system, namely complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Unfortunately, in some patients the resistance to anti-CD20 therapy has been detected. Therefore the molecular mechanisms responsible for the therapy failure should be urgently elucidated. One of the major mechanisms responsible for the resistance to anti-CD20 therapy seems to be the reduced level of CD20 antigen on the surface of tumor B-cells. We have previously discovered that CD20 expression is strictly dependent on the activity of Src family tyrosine kinases and AKT kinase. We have noticed that treatment of B cells with Src family inhibitors or AKT inhibitors leads to a dose-dependent reduction of CD20 at both transcript and protein level. On the other hand the overexpression of constitutively active AKT (CA-AKT) leads to a significant increase in CD20 mRNA level. To uncover the transcriptional mechanisms governing the CD20 expression we employed the construct encoding the promoter region of CD20 cloned upstream of the firefly luciferase gene. The truncated and mutated versions of the CD20 promoter were used in the luciferase assays to elucidate the role of particular transcription factors binding sites in the regulation of CD20 expression. Objectives The aim of this study was to explore the molecular mechanisms governing the transcriptional regulation of CD20 expression in lymphoma cells as a potential explanation of the resistance to anti-CD20 therapy. Results In the initial experiments we observed a significant reduction of CD20 protein level in Raji cells treated with Src family tyrosine kinase inhibitor, dasatinib (Fig. 1A,B). This reduction correlated with the impaired binding of anti-CD20 monoclonal antibodies as estimated by FACS analysis. Quantitative PCR analysis revealed that the transcriptional regulation is the major mechanism responsible for the reduction of CD20 level upon dasatinib treatment (Fig. 1C). Consistently, the exogenously expressed CD20 under the control of CMV promoter was not sensitive to dasatinib treatment. To further elucidate the mechanism of transcriptional regulation of CD20 we performed the luciferase assays to estimate the activity of CD20 promoter and its truncated forms (Fig. 1D). Dasatinib or AKT kinase inhibitor (MK-2206) strongly decreased the activity of CD20 promoter (Fig. 1E,F) while the overexpression of CA-AKT partially blocked the inhibition caused by dasatinib (Fig. 1G). Using the truncated versions of the CD20 promoter we found that lack of the region (-313/-198) made the promoter insensitive to dasatinib treatment. Since this particular region is known to contain a putative Octamer transcription factor binding site (BAT-box, Thevenin et al., 1993), we introduced mutations in the BAT-box sequence. Although basal promoter activity was indeed decreased (Fig. 1H), dasatinib was equally effective in reducing the activity of both wild-type and mutated CD20 promoter. Collectively, our results indicate that Octamer transcription factor is an important regulator of basal CD20 expression, but it is not the major mediator of the effects caused by Src family inhibitors. Conclusions Our studies indicate that the Src family tyrosine kinases and AKT kinase are involved in the transcriptional regulation of CD20 antigen in lymphoma cells. The activity of CD20 promoter is significantly reduced upon treatment with Src family inhibitors, namely dasatinib. The particular region of CD20 promoter (-313/-198) was identified as the major region sensitive to dasatinib treatment. The transcriptional machinery responsible for the reduction of CD20 expression by dasatinib needs further investigation since the expected Octamer transcription factor does not mediate the effects caused by dasatinib. Disclosures: No relevant conflicts of interest to declare.

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