cJun Is Phosphorylated at Ser73 and Contributes to Cell Cycle Progression in Anaplastic Large Cell Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2620-2620
Author(s):  
Vassiliki Leventaki ◽  
Elias Drakos ◽  
Francois-Xavier Claret ◽  
L. Jeffrey Medeiros ◽  
George Z. Rassidakis
Keyword(s):  
Cell Cycle ◽  
Tumor Cells ◽  
Cell Lines ◽  
Anaplastic Large Cell Lymphoma ◽  
Cell Cycle Progression ◽  
Cell Lymphoma ◽  
Large Cell ◽  
S Phase ◽  
Cycle Progression ◽  
Large Cell Lymphoma

Abstract Anaplastic Large Cell Lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). cJun is a member of the activator protein-1 (AP-1) family, which is a group of transcription factors that control cell proliferation, differentiation, growth and apoptosis. The activity of cJun can be regulated by phosphorylation at serine 73 (Ser73) and serine 63 (Ser63) residues of the N-terminal domain. It is believed that cJun promotes cell cycle progression, in part, through downregulation of the cyclin-dependent kinase inhibitor p21. Previous studies have shown high AP-1 activity and cJun overexpression in Hodgkin lymphoma and ALCL (Mathas et al, EMBO J2002; 21:4104). In this study, we assessed for expression of cJun and its Ser73- and Ser63-phosphorylated forms in two ALK+ (Karpas 299 and SU-DHL-1) and one ALK- (Mac2A) ALCL cell lines by western blot analysis, and in 31 ALCL tumors (15 ALK+, 16 ALK-) by immunohistochemistry using tissue microarrays and specific antibodies. To examine the role of cJun in cell survival and proliferation in our in vitro system, ALCL cells were transiently transfected with small interfering RNA (siRNA) specific for cJun. Cell viability, proliferation of viable cells and cell cycle progression from G1 to S-phase were assessed by trypan blue exclusion, MTS and BrdU assays, respectively. All three ALCL cell lines expressed total cJun and Ser73-phosphorylated cJun (Ser73p-cJun) at a high level, whereas Ser63-phosphorylated cJun was expressed at a low level. In addition, all 31 ALCL tumors expressed total cJun in most neoplastic cells. Ser73p-cJun was also detected in all ALCL tumors at a variable level with the percentage of Ser73p-cJun-positive tumor cells ranging from 5% to 95%. By contrast, Ser63p-cJun was detected rarely in tumor cells. Transient transfection of ALCL cells with specific siRNA resulted in almost complete silencing of total cJun expression and absence of Ser73p-cJun expression, which was associated with decreased cell viability and a substantial (40%) decrease of cell growth. cJun silencing also resulted in cell cycle arrest as shown by decreased S-phase fraction. These cell cycle changes were associated with a marked increase of p21 levels and downregulation of cyclin D2 and D3. In conclusion, cJun is highly phosphorylated at serine 73 in ALCL cell lines and tumors and may contribute to cell cycle progression. Targeting cJun expression or phosphorylation using gene therapy approaches may represent a novel therapeutic strategy for patients with ALCL.

NPM-ALK Promotes Cell Cycle Progression through Activation of JNK and c-Jun in Anaplastic Large Cell Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2057-2057
Author(s):  
Vasiliki Leventaki ◽  
Elias Drakos ◽  
Megan Lim ◽  
Kojo S. Elenitoba-Johnson ◽  
Francois-Xavier Claret ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Western Blot ◽  
Anaplastic Large Cell Lymphoma ◽  
Blot Analysis ◽  
Cell Cycle Progression ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Cycle Progression ◽  
Large Cell Lymphoma

Abstract Anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) chimeric protein. NPM-ALK mediates its oncogenic effects through phosphorylation of a number of proteins involved in known signal transduction pathways including PLC, PI3K-AKT and JAK-STAT. ALK+ ALCL cells also are known to overexpress c-Jun, a member of the activator protein-1 (AP-1) transcription factor family that controls cell proliferation, differentiation, growth and apoptosis. Phosphorylation of c-Jun at serine 73 and serine 63 residues substantially increases AP-1 transcriptional activity and the levels of c-Jun protein through an autoregulatory positive feedback loop. In this study, we hypothesized that NPM-ALK activates JNK which , in turn, phosphorylates and activates c-Jun, resulting in uncontrolled cell cycle progression in ALCL. 293T and Jurkat (T-acute lymphoblastic leukemia) cells were transfected with a vector expressing NPM-ALK with active kinase domain (pDest40-NPM-ALK) or a construct lacking NPM-ALK kinase activity (pDest40-K210R) or empty vector. Cells were harvested at 48 hours and analyzed for protein expression by Western blot analysis and for AP-1 activity by luciferase reporter assay. Two ALK+ ALCL cell lines Karpas 299 and SU-DHL-1, found to express high levels of serine phosphorylated and total c-Jun in immunoblots, were treated with JNK (SP600125), ERK (U0126), or ALK (WHI-P154) inhibitors or were transiently transfected with siRNAs specific for JNK1 and c-Jun. Cell proliferation was assessed by MTS assay, and cell cycle was analyzed by BrdU assay or propidium iodide staining and flow cytometry. Forced expression of NPM-ALK in 293T and Jurkat cells resulted in increased levels of JNK and c-Jun phosphorylation in immunoblots and a dramatic increase in AP-1 activity. Conversely, pharmacologic inhibition of ALK activity in Karpas 299 and SU-DHL1 resulted in a concentration-dependent decrease of JNK and c-Jun phosphorylation levels. Co-immunoprecipitation studies revealed that NPM-ALK physically binds to JNK1 and its upstream activator MKK7 in ALK+ ALCL cells. Selective inhibition of JNK, but not ERK, in Karpas 299 and SU-DHL1 decreased the level of c-Jun phosphorylation in a dose-dependent manner as shown by Western blot analysis and in vitro kinase assays. Inhibition of JNK by SP600125 or silencing of the JNK1 gene by siRNA also resulted in decreased cell proliferation associated with decreased AP-1 activity, cell cycle arrest mostly at G2 phase, and up-regulation of the cyclin-dependent inhibitor p21, a transcriptional target of c-Jun. Similarly, silencing of c-Jun by specific siRNA led to decreased S-phase fraction of cell cycle, which was associated with up-regulation of p21 and downregulation of cyclin D3. These findings reveal a novel function of NPM-ALK oncoprotein, phosphorylation and activation of JNK, which may contribute to uncontrolled cell cycle progression through activation of c-Jun. Modulation of JNK or c-Jun activity may be a target for therapy in patients with ALCL.

NPM-ALK oncogenic kinase promotes cell-cycle progression through activation of JNK/cJun signaling in anaplastic large-cell lymphoma

Blood ◽  
2007 ◽  
Vol 110 (5) ◽  
pp. 1621-1630 ◽  
Author(s):  
Vasiliki Leventaki ◽  
Elias Drakos ◽  
L. Jeffrey Medeiros ◽  
Megan S. Lim ◽  
Kojo S. Elenitoba-Johnson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Anaplastic Large Cell Lymphoma ◽  
Cell Cycle Progression ◽  
Transcriptional Activity ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Cyclin D3 ◽  
Phase Cell ◽  
Cycle Progression ◽  
Large Cell Lymphoma

Abstract Anaplastic large-cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35), resulting in aberrant expression of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). We show that in 293T and Jurkat cells, forced expression of active NPM-ALK, but not kinase-dead mutant NPM-ALK (210K>R), induced JNK and cJun phosphorylation, and this was linked to a dramatic increase in AP-1 transcriptional activity. Conversely, inhibition of ALK activity in NPM-ALK+ ALCL cells resulted in a concentration-dependent dephosphorylation of JNK and cJun and decreased AP-1 DNA-binding. In addition, JNK physically binds NPM-ALK and is highly activated in cultured and primary NPM-ALK+ ALCL cells. cJun phosphorylation in NPM-ALK+ ALCL cells is mediated by JNKs, as shown by selective knocking down of JNK1 and JNK2 genes using siRNA. Inhibition of JNK activity using SP600125 decreased cJun phosphorylation and AP-1 transcriptional activity and this was associated with decreased cell proliferation and G2/M cell-cycle arrest in a dose-dependent manner. Silencing of the cJun gene by siRNA led to a decreased S-phase cell-cycle fraction associated with upregulation of p21 and downregulation of cyclin D3 and cyclin A. Taken together, these findings reveal a novel function of NPM-ALK, phosphorylation and activation of JNK and cJun, which may contribute to uncontrolled cell-cycle progression and oncogenesis.

scholarly journals Inhibition of Akt increases p27Kip1 levels and induces cell cycle arrest in anaplastic large cell lymphoma

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 827-829 ◽  
Author(s):  
George Z. Rassidakis ◽  
Marianna Feretzaki ◽  
Coralyn Atwell ◽  
Ioannis Grammatikakis ◽  
Quan Lin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Anaplastic Large Cell Lymphoma ◽  
Cell Cycle Progression ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Cycle Progression ◽  
Anaplastic Lymphoma ◽  
Cycle Arrest ◽  
Large Cell Lymphoma

Abstract Anaplastic large cell lymphoma (ALCL) is a highly proliferative neoplasm that frequently carries the t(2;5)(p23;q35) and aberrantly expresses nucleophosmin–anaplastic lymphoma kinase (NPM-ALK). Previously, NPM-ALK had been shown to activate the phosphatidylinositol 3 kinase (PI3K)/Akt pathway. As the cyclin-dependent kinase (CDK) inhibitor p27Kip1 (p27) is usually not expressed in ALCL, we hypothesized that activated Akt (pAkt) phosphorylates p27 resulting in increased p27 proteolysis and cell cycle progression. Here we demonstrate that inhibition of pAkt activity in ALCL decreases p27 phosphorylation and degradation, resulting in increased p27 levels and cell cycle arrest. Using immunohistochemistry, pAkt was detected in 24 (57%) of 42 ALCL tumors, including 8 (44%) of 18 ALK-positive tumors and 16 (67%) of 24 ALK-negative tumors, and was inversely correlated with p27 levels. The mean percentage of p27-positive tumor cells was 5% in the pAkt-positive group compared with 26% in the pAkt-negative group (P = .0076). These findings implicate that Akt activation promotes cell cycle progression through inactivation of p27 in ALCL.

Activation of mTOR Signaling Pathway Contributes to Tumoral Cell Survival in ALK-Positive Anaplastic Large Cell Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2419-2419
Author(s):  
Francisco Vega ◽  
L. Jeffrey Medeiros ◽  
Coralyn Atwell ◽  
Jeong Hee Cho ◽  
Ling Tian ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Anaplastic Large Cell Lymphoma ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Sirna Treatment ◽  
Cycle Arrest ◽  
Large Cell Lymphoma ◽  
Alk Positive

Abstract Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of nucleophosmin (NPM)-ALK. Previously, NPM-ALK has been shown to activate phosphatidylinositol 3-kinase (PI3K) and its downstream effector, the serine/threonine kinase AKT. Recently, we have shown that mTOR signaling proteins are activated in ALK-positive ALCL tumors and that mTOR activation depends, at least in part, on activation of AKT (Lab Invest2005; 85: 255A). In this study, we investigate the biological effects of inhibition of mTOR on two ALK-positive ALCL cell lines, Karpas 299 and SU-DHL1. For this purpose, we used rapamycin to inhibit mTOR-raptor complex and mTOR-specific small interfering RNA (siRNA) to silence the endogenous mtor gene. Treatment with rapamycin, resulted in a marked concentration-dependent decrease of phosphorylated (p)-mTOR, and its downstream targets, p-p70S6K, p-S6K, p-4E-BP1 and total eIF4E. Similarly, silencing the expression of mtor resulted in a decrease in the activation/phosphorylation level of these proteins as well as in the level of p-AKT. Both treatments induced apoptosis and cell cycle arrest in both ALK-positive ALCL cell lines as demonstrated by trypan blue exclusion, annexin V staining, BrdU incorporation, and cell cycle studies. There was a concentration-dependent decrease in the anti-apoptotic proteins BCL-2, BCL-XL, MCL-1 and c-FLIP (L and S) with increasing concentrations of rapamycin or after mTOR siRNA treatment. The cyclin dependent kinase inhibitors p21waf1 and p27kip1 and underphosphorylated (Un-p)-RB protein were upregulated, after treatment with rapamycin or after mTOR siRNA treatment. In conclusion, we provide evidence that inhibition of mTOR induces cell cycle arrest and apoptosis in ALK-positive ALCL cells. The decrease of p-AKT by silencing mtor suggests that mTOR is necessary to activate AKT in ALK-positive ALCL, and thus, mTOR can function as a feedback signal activity of its own pathway.

scholarly journals Autocrine release of interleukin-9 promotes Jak3-dependent survival of ALK+ anaplastic large-cell lymphoma cells

Blood ◽  
2006 ◽  
Vol 108 (7) ◽  
pp. 2407-2415 ◽  
Author(s):  
Lin Qiu ◽  
Raymond Lai ◽  
Quan Lin ◽  
Esther Lau ◽  
David M. Thomazy ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Anaplastic Large Cell Lymphoma ◽  
Kinase Activity ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Primary Tumors ◽  
Cycle Arrest ◽  
Large Cell Lymphoma

Abstract The aberrant fusion protein NPM-ALK plays an important pathogenetic role in ALK+ anaplastic large-cell lymphoma (ALCL). We previously demonstrated that Jak3 potentiates the activity of NPM-ALK. Jak3 activation is restricted to interleukins that recruit the common γ chain (γc) receptor, including IL-9. NPM-ALK was previously shown to promote widespread lymphomas in IL-9 transgenic mice by unknown mechanisms. We hypothesized that IL-9 plays an important role in ALK+ ALCL via Jak3 activation. Our studies demonstrate the expression of IL-9Rα and IL-9 in 3 ALK+ ALCL-cell lines and 75% and 83% of primary tumors, respectively. IL-9 was detected in serum-free culture medium harvested from ALK+ ALCL-cell lines, supporting autocrine release of IL-9. Treatment of these cells with an anti–IL-9–neutralizing antibody decreased pJak3 and its kinase activity, along with pStat3 and ALK kinase activity. These effects were associated with decreased cell proliferation and colony formation in soft agar and cell-cycle arrest. Evidence suggests that cell-cycle arrest can be attributed to up-regulation of p21 and down-regulation of Pim-1. Our results illustrate that IL-9/Jak3 signaling plays a significant role in the pathogenesis of ALK+ ALCL and that it represents a potential therapeutic target for treating patients with ALK+ ALCL.

scholarly journals Gram-Negative Bacterial Lipopolysaccharide Promotes Tumor Cell Proliferation in Breast Implant-Associated Anaplastic Large-Cell Lymphoma

Cancers ◽  
2021 ◽  
Vol 13 (21) ◽  
pp. 5298
Author(s):  
Maria Mempin ◽  
Honghua Hu ◽  
Karen Vickery ◽  
Marshall E. Kadin ◽  
H. Miles Prince ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Cells ◽  
Cell Lines ◽  
Anaplastic Large Cell Lymphoma ◽  
Cell Lymphoma ◽  
Breast Implant ◽  
Large Cell ◽  
Bacterial Lipopolysaccharide ◽  
Gram Negative ◽  
Large Cell Lymphoma

Breast implant-associated anaplastic large-cell lymphoma (BIA-ALCL) is a distinct malignancy associated with textured breast implants. We investigated whether bacteria could trigger the activation and multiplication of BIA-ALCL cells in vitro. BIA-ALCL patient-derived BIA-ALCL tumor cells, BIA-ALCL cell lines, cutaneous ALCL cell lines, an immortal T-cell line (MT-4), and peripheral blood mononuclear cells (PBMC) from BIA-ALCL, capsular contracture, and primary augmentation patients were studied. Cells were subjected to various mitogenic stimulation assays including plant phytohemagglutinin (PHA), Gram-negative bacterial lipopolysaccharide (LPS), Staphylococcal superantigens enterotoxin A (SEA), toxic shock syndrome toxin-1 (TSST-1), or sterilized implant shells. Patient-derived BIA-ALCL tumor cells and BIA-ALCL cell lines showed a unique response to LPS stimulation. This response was dampened significantly in the presence of a Toll-like receptor 4 (TLR4) inhibitor peptide. In contrast, cutaneous ALCL cells, MT-4, and PBMC cells from all patients responded significantly more to PHA, SEA, and TSST-1 than to LPS. Breast implant shells of all surface grades alone did not produce a proliferative response of BIA-ALCL cells, indicating the breast implant does not act as a pro-inflammatory stimulant. These findings indicate a possible novel pathway for LPS to promote BIA-ALCL cell proliferation via a TLR4 receptor-mediated bacterial transformation of T-cells into malignancy.

scholarly journals Migration Properties Distinguish Tumor Cells of Classical Hodgkin Lymphoma from Anaplastic Large Cell Lymphoma Cells

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1484
Author(s):  
Olga Goncharova ◽  
Nadine Flinner ◽  
Julia Bein ◽  
Claudia Döring ◽  
Emmanuel Donnadieu ◽  
...  
Keyword(s):  
Cell Motility ◽  
Hodgkin Lymphoma ◽  
Tumor Cells ◽  
Cell Lines ◽  
Anaplastic Large Cell Lymphoma ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Receptor Expression ◽  
Classical Hodgkin Lymphoma ◽  
Large Cell Lymphoma

Anaplastic large cell lymphoma (ALCL) and classical Hodgkin lymphoma (cHL) are lymphomas that contain CD30-expressing tumor cells and have numerous pathological similarities. Whereas ALCL is usually diagnosed at an advanced stage, cHL more frequently presents with localized disease. The aim of the present study was to elucidate the mechanisms underlying the different clinical presentation of ALCL and cHL. Chemokine and chemokine receptor expression were similar in primary ALCL and cHL cases apart from the known overexpression of the chemokines CCL17 and CCL22 in the Hodgkin and Reed-Sternberg (HRS) cells of cHL. Consistent with the overexpression of these chemokines, primary cHL cases encountered a significantly denser T cell microenvironment than ALCL. Additionally to differences in the interaction with their microenvironment, cHL cell lines presented a lower and less efficient intrinsic cell motility than ALCL cell lines, as assessed by time-lapse microscopy in a collagen gel and transwell migration assays. We thus propose that the combination of impaired basal cell motility and differences in the interaction with the microenvironment hamper the dissemination of HRS cells in cHL when compared with the tumor cells of ALCL.

scholarly journals Everolimus in Combination with Crizotinib Synergistically Inhibits the Growth of ALK-Positive Anaplastic Large Cell Lymphoma Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4488-4488
Author(s):  
Wendan Xu ◽  
Ji-Won Kim ◽  
Junglim Lee ◽  
Hyo Jung Kim ◽  
Hwi-Joong Yoon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Combination Treatment ◽  
Anaplastic Large Cell Lymphoma ◽  
Fusion Gene ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Large Cell Lymphoma ◽  
Alk Positive

Abstract More than a half of anaplastic large cell lymphoma (ALCL) harbors an aberrant NPM-ALK fusion gene, which activates a number of down-stream signaling pathways such as Ras/ERK, PI3K/AKT, and JAK3/STAT3. Through this mechanism, mTOR pathway is also activated in ALK-positive ALCL (Vega F, et al. Cancer Res 2006). Everolimus, an mTOR inhibitor, has shown promising anti-tumor activity in a variety of lymphomas (Jundt F, et al. Blood 2005; Wanner K, et al. Br J Haematol 2006; Haritunians T, et al. Leukemia 2007), although the clinical efficacy of everolimus monotherapy was not satisfactory, possibly due to activation of several pro-surviving signaling pathways. The combined effect of everolimus and crizotinib, an ALK inhibitor, has not yet been investigated in ALK-positive tumors so far. The aim of this study was to evaluate the effect of everolimus in combination with crizotinib in ALK-positive ALCL cell lines, K-299 and SU-DHL-1. We treated K-299 and SU-DHL-1 cells with various concentrations of everolimus and crizotinib at a fixed ratio of 1:40 (Figure 1). After 72 hours, the combination index (CI) values calculated by the Chou-Talalay method were less than 1 (range, 0.583-0.763 in K-299 cells and 0.271-0.616 in SU-DHL-1 cells) in all tested combinations, suggesting synergistic cytotoxicity of everolimus and crizotinib. The Western blot analysis (Figure 2) demonstrated that everolimus treatment up-regulated the phosphorylation of ERK Thr202/Tyr204 and AKT Thr308 and Ser473 in K-299 cells. However, this aberrant activation of ERK and AKT was attenuated by the addition of crizotinib. In addition, while everolimus selectively inhibited phosphorylation of mTOR Ser2448, a marker for mTORC1 activity, the combination treatment more potently inhibited mTOR Ser2448 phosphorylation and decreased phosphorylated mTOR at Ser2481, a marker for mTORC2, as well. In the cell-cycle analysis, the combination treatment induced G1 arrest. Everolimus treatment alone did not increase the fraction of cells in the sub-G1 region compared to the control (2.16% vs. 4.03% in K-299 and 1.34% vs. 1.68% in SU-DHL-1), while crizotinib monotherapy increased the sub-G1 population (11.88% vs. 4.03% in K-299 and 28.68% vs. 1.68% in SU-DHL-1). The combination of crizotinib and everolimus markedly increased the sub-G1 population in both ALK-positive ALCL cell lines (22.25% in K-299 and 46.40% in SU-DHL-1). PARP cleavage was also increased after the combination treatment. To test the hypothesis that our findings could be applyed to other ALK-positive malignancies, we treated NCI-H2228, a lung adenocarcinoma cell line that harbors an EML4-ALK fusion gene, with everolimus and crizotinib for 72 hours. The CI values were less than 1 in all tested combinations: 0.228 in 1 nM everolimus plus 80 nM crizotinib, 0.216 in 2 nM everolimus plus 160 nM crizotinib, and 0.349 in 4 nM everolimus and 320 nM crizotinib. In summary, everolimus combined with crizotinib synergistically inhibited the growth of ALK-positive ALCL cells. Crizotinib abrogated aberrant ERK and AKT signaling activation induced by everolimus and more potently inhibited both mTORC1 and mTORC2 activity when combined with everolimus, resulting in increased G1 cell-cycle arrest and apoptosis (Figure 3). Our findings may provide an evidence for future research using everolimus and crizotinib combination in ALK-positive ALCL and could be used to improve the therapeutic outcome in patients with ALK-positive ALCL. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

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