@prefix rdf: . @prefix ns1: . rdf:type ns1:RelevantPaper , ns1:ReferencePaper , ns1:CitationPaper . @prefix rdfs: . rdfs:seeAlso , , , . @prefix bibo: . bibo:cites , , , , , , , , , , , , , , , , , , ; ns1:cocitationWith , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ; ns1:hasRelevantBibliographicResourceOf _:b447415680 , _:b447415681 , _:b447415682 , _:b447415683 , _:b447415684 , _:b447415685 , _:b447415686 , _:b447415687 , _:b447415688 , _:b447415689 , _:b447415690 , _:b447415691 , _:b447415692 , _:b447415693 , _:b447415694 , _:b447415695 , _:b447415696 , _:b447415697 , _:b447415698 , _:b447415699 , _:b447415700 , _:b447415701 , _:b447415702 , _:b447415703 , _:b447415704 , _:b447415705 , _:b447415706 , _:b447415707 , _:b447415611 , _:b447415612 , _:b447415613 , _:b447415614 , _:b447415615 , _:b447415616 , _:b447415617 , _:b447415618 , _:b447415619 , _:b447415620 , _:b447415621 , _:b447415622 , _:b447415623 , _:b447415624 , _:b447415625 , _:b447415626 , _:b447415627 , _:b447415628 , _:b447415629 , _:b447415630 , _:b447415631 , _:b447415632 , _:b447415633 , _:b447415634 , _:b447415635 , _:b447415636 , _:b447415637 , _:b447415638 , _:b447415639 , _:b447415640 , _:b447415641 , _:b447415642 , _:b447415643 , _:b447415644 , _:b447415645 , _:b447415646 , _:b447415647 , _:b447415648 , _:b447415649 , _:b447415650 , _:b447415651 , _:b447415652 , _:b447415653 , _:b447415654 , _:b447415655 , _:b447415656 , _:b447415657 , _:b447415658 , _:b447415659 , _:b447415660 , _:b447415661 , _:b447415662 , _:b447415663 , _:b447415664 , _:b447415665 , _:b447415666 , _:b447415667 , _:b447415668 , _:b447415669 , _:b447415670 , _:b447415671 , _:b447415672 , _:b447415673 , _:b447415674 , _:b447415675 , _:b447415676 , _:b447415677 , _:b447415678 , _:b447415679 ; ns1:pmcid "PMC0" ; bibo:doi "10.1186%2F1476-4598-9-292" . @prefix ns4: . ns4:contains _:b9238580 , _:b9238562 , _:b9238569 . _:b9238562 rdf:type ns4:Section . @prefix dc: . _:b9238562 dc:title "methods" ; ns4:contains _:b9238568 , _:b9238564 , _:b9238565 , _:b9238566 , _:b9238567 , _:b9238563 . _:b9238563 rdf:type ns1:Context ; rdf:value "Three human AML cell lines were used; MOLM-13 (heterozygous for FLT3-ITD), MV4-11 (homozygous for FLT3-ITD) [>>18<<], U937 (wild-type FLT3), and murine BaF3 cells transfected with wild-type (Wt) FLT3 (BaF3/FLT3) or mutated FLT3 (FLT3/ITD and FLT3/D835G) were cultured under standard conditions as previously described [18]." ; ns1:mentions . _:b9238564 rdf:type ns1:Context ; rdf:value "(homozygous for FLT3-ITD) [18], U937 (wild-type FLT3), and murine BaF3 cells transfected with wild-type (Wt) FLT3 (BaF3/FLT3) or mutated FLT3 (FLT3/ITD and FLT3/D835G) were cultured under standard conditions as previously described [>>18<<]. The murine cell line BaF3 was kindly provided by Dr. M Andreeff (M.D. Anderson Cancer Center, Houston, TX)." ; ns1:mentions . _:b9238565 rdf:type ns1:Context ; rdf:value "Immunohistochemistry was performed using histologic sections of the tissue microarray or cell blocks, and antibodies specific for p-mTOR, p-p70S6K, p-rpS6, p-4E-BP1, and eIF4E (Cell Signaling Technology) as previously described [>>19<<]. AML cases were considered immunohistochemically positive for any of the proteins analyzed if at least 10% of tumor cells showed expression after examination of 10 representative high-power fields (magnification \u00D7400)." ; ns1:mentions . _:b9238566 rdf:type ns1:Context ; rdf:value "Western blot analysis was performed as previously described[>>19<<]. The antibodies used included Ser473pAKT (p-pAKT), total Akt, p-mTOR, p-p70S6K, p70S6K, p-rpS6, rpS6, p4E-BP1, 4E-BP1, eIF4E (Cell Signaling Technology, Beverly, MA), and \u03B2-actin (Sigma, St." ; ns1:mentions . _:b9238567 rdf:type ns1:Context ; rdf:value "Annexin-V staining (BD Biosciences Pharmingen, San Diego, CA), detected by flow cytometry, was used to assess apoptosis, as previously described [>>20<<]." ; ns1:mentions . _:b9238568 rdf:type ns1:Context ; rdf:value "Proliferation of the cells was assessed by using a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, MTS] (Promega, Madison, WI, USA), as previously described [>>20<<]. All experiments were performed at least twice." ; ns1:mentions . _:b9238569 rdf:type ns4:Section ; dc:title "background" ; ns4:contains _:b9238570 , _:b9238571 , _:b9238572 , _:b9238573 , _:b9238574 , _:b9238575 , _:b9238576 , _:b9238577 , _:b9238578 , _:b9238579 . _:b9238570 rdf:type ns1:Context ; rdf:value "have shown that constitutive activation of FLT-3 is involved in leukemogenesis, partially through phosphorylation/activation of the serine-threonine kinase AKT, which occurs in a substantial subset of acute myeloid leukemia (AML) cases [>>1<<,2]. A frequent mechanism of FLT3 activation is mutation of the FLT3 gene, either internal tandem duplication (ITD) within the juxtamembrane domain (FLT3-ITD), or point mutations within the activation loop of the second tyrosine kinase" ; ns1:mentions . _:b9238571 rdf:type ns1:Context ; rdf:value "shown that constitutive activation of FLT-3 is involved in leukemogenesis, partially through phosphorylation/activation of the serine-threonine kinase AKT, which occurs in a substantial subset of acute myeloid leukemia (AML) cases [1,>>2<<]. A frequent mechanism of FLT3 activation is mutation of the FLT3 gene, either internal tandem duplication (ITD) within the juxtamembrane domain (FLT3-ITD), or point mutations within the activation loop of the second tyrosine kinase" ; ns1:mentions . _:b9238572 rdf:type ns1:Context ; rdf:value "The mammalian target of rapamycin (mTOR), an important downstream effector of AKT, is a master regulator of cell growth and metabolism [>>4<<]. There are two mTOR multi-protein complexes, mTOR-Raptor/mTORC1 and mTOR-Rictor/mTORC2." ; ns1:mentions . _:b9238573 rdf:type ns1:Context ; rdf:value "mTOR-Raptor/mTORC1 is sensitive to the natural macrolide rapamycin and regulates the rate of protein translation [>>4<<,5]. This regulation is accomplished, partially, through phosphorylation of the ribsosomal protein S6 kinase (p70S6K) and subsequent phosphorylation of the ribosomal protein S6 (rpS6), or phosphorylation and inactivation of the eukaryotic" ; ns1:mentions . _:b9238574 rdf:type ns1:Context ; rdf:value "mTOR-Raptor/mTORC1 is sensitive to the natural macrolide rapamycin and regulates the rate of protein translation [4,>>5<<]. This regulation is accomplished, partially, through phosphorylation of the ribsosomal protein S6 kinase (p70S6K) and subsequent phosphorylation of the ribosomal protein S6 (rpS6), or phosphorylation and inactivation of the eukaryotic" ; ns1:mentions . _:b9238575 rdf:type ns1:Context ; rdf:value "S6 (rpS6), or phosphorylation and inactivation of the eukaryotic initiation factor 4E (eIF4E)-binding protein-1 (4E-BP1), dissociating 4E-BP1 from the RNA cap-binding protein eIF4E, thus promoting cap-dependent translation of mRNAs [>>4<<,5]. mTOR-Rictor/mTORC2, usually insensitive to rapamycin, phosphorylates AKT at serine residue 473, contributing to AKT activation, and establishing an autoregulatory loop [6]." ; ns1:mentions . _:b9238576 rdf:type ns1:Context ; rdf:value "S6 (rpS6), or phosphorylation and inactivation of the eukaryotic initiation factor 4E (eIF4E)-binding protein-1 (4E-BP1), dissociating 4E-BP1 from the RNA cap-binding protein eIF4E, thus promoting cap-dependent translation of mRNAs [4,>>5<<]. mTOR-Rictor/mTORC2, usually insensitive to rapamycin, phosphorylates AKT at serine residue 473, contributing to AKT activation, and establishing an autoregulatory loop [6]." ; ns1:mentions . _:b9238577 rdf:type ns1:Context ; rdf:value "mTOR-Rictor/mTORC2, usually insensitive to rapamycin, phosphorylates AKT at serine residue 473, contributing to AKT activation, and establishing an autoregulatory loop [>>6<<]." ; ns1:mentions . _:b9238578 rdf:type ns1:Context ; rdf:value "Recent studies have shown that rapamycin, and its analogs, have substantial antitumor activity in hematologic malignancies, including AML [>>7<<,8]. However, the significance of FLT3 gene mutation in the activation of the mTOR pathway is not clear." ; ns1:mentions . _:b9238579 rdf:type ns1:Context ; rdf:value "Recent studies have shown that rapamycin, and its analogs, have substantial antitumor activity in hematologic malignancies, including AML [7,>>8<<]. However, the significance of FLT3 gene mutation in the activation of the mTOR pathway is not clear." ; ns1:mentions . _:b9238580 rdf:type ns4:Section ; dc:title "results and discussion" ; ns4:contains _:b9238581 , _:b9238582 , _:b9238583 , _:b9238588 , _:b9238589 , _:b9238590 , _:b9238591 , _:b9238584 , _:b9238585 , _:b9238586 , _:b9238587 , _:b9238592 . _:b9238581 rdf:type ns1:Context ; rdf:value "We also showed activated mTOR signaling by Western blot analysis (Figure 1), in agreement with others [>>9<<]. To further investigate a possible causal association between FLT3 mutation and the mTOR activation status, we used stably transfected FLT3-mutant BaF3 cells. Introduction of FLT3-ITD in BaF3 cells resulted in activation of mTOR" ; ns1:mentions . _:b9238582 rdf:type ns1:Context ; rdf:value "Most previous studies investigating this issue were based on rapamycin treatment of AML cells resulting frequently in conflicting conclusions [>>10<<,11]. However, recently it was realized that rapamycin not only can indirectly and unpredictably inhibit the rapamycin-insensitive mTORC2 after prolonged treatment, but also frequently inhibits partially and not totally mTORC1, which is" ; ns1:mentions . _:b9238583 rdf:type ns1:Context ; rdf:value "Most previous studies investigating this issue were based on rapamycin treatment of AML cells resulting frequently in conflicting conclusions [10,>>11<<]. However, recently it was realized that rapamycin not only can indirectly and unpredictably inhibit the rapamycin-insensitive mTORC2 after prolonged treatment, but also frequently inhibits partially and not totally mTORC1, which is" ; ns1:mentions . _:b9238584 rdf:type ns1:Context ; rdf:value "For this reason, we employed the pharmacologic PI3K inhibitor LY294002, known to inhibit both the PI3K and mTOR kinase activity with the same kinetics, or total silencing of the mTOR gene using an mTOR-specific siRNA [>>12<<,13]." ; ns1:mentions . _:b9238585 rdf:type ns1:Context ; rdf:value "For this reason, we employed the pharmacologic PI3K inhibitor LY294002, known to inhibit both the PI3K and mTOR kinase activity with the same kinetics, or total silencing of the mTOR gene using an mTOR-specific siRNA [12,>>13<<]." ; ns1:mentions . _:b9238586 rdf:type ns1:Context ; rdf:value "Taken together, our findings from mTOR gene silencing and pharmacologic studies suggest that mTOR signaling may contribute to the survival of AML cells harboring FLT3 mutations, and are concordant with the results of other studies [>>8<<,14,15]." ; ns1:mentions . _:b9238587 rdf:type ns1:Context ; rdf:value "Taken together, our findings from mTOR gene silencing and pharmacologic studies suggest that mTOR signaling may contribute to the survival of AML cells harboring FLT3 mutations, and are concordant with the results of other studies [8,>>14<<,15]." ; ns1:mentions . _:b9238588 rdf:type ns1:Context ; rdf:value "Taken together, our findings from mTOR gene silencing and pharmacologic studies suggest that mTOR signaling may contribute to the survival of AML cells harboring FLT3 mutations, and are concordant with the results of other studies [8,14,>>15<<]." ; ns1:mentions . _:b9238589 rdf:type ns1:Context ; rdf:value "evidence that mTOR signaling is an important downstream mediator of the oncogenic pathway activated by mutated FLT3 kinase (Figure 3), and that direct inhibition of mTOR may possibly augment the therapeutic potential of FLT3 inhibitors[>>16<<]. Our findings are in agreement with previous observations that rapamycin in combination with the FLT3 inhibitor, PKC-412, markedly inhibits cell proliferation in murine BaF-FLT3-ITD cells and AML samples bearing FLT3 gene dual mutations" ; ns1:mentions . _:b9238590 rdf:type ns1:Context ; rdf:value "PKC-412, markedly inhibits cell proliferation in murine BaF-FLT3-ITD cells and AML samples bearing FLT3 gene dual mutations of ITD and point mutation types, and that rapamycin inhibits cell proliferation in murine 32D-FLT3-ITD cells [>>2<<,8,17]." ; ns1:mentions . _:b9238591 rdf:type ns1:Context ; rdf:value "PKC-412, markedly inhibits cell proliferation in murine BaF-FLT3-ITD cells and AML samples bearing FLT3 gene dual mutations of ITD and point mutation types, and that rapamycin inhibits cell proliferation in murine 32D-FLT3-ITD cells [2,>>8<<,17]." ; ns1:mentions . _:b9238592 rdf:type ns1:Context ; rdf:value "markedly inhibits cell proliferation in murine BaF-FLT3-ITD cells and AML samples bearing FLT3 gene dual mutations of ITD and point mutation types, and that rapamycin inhibits cell proliferation in murine 32D-FLT3-ITD cells [2,8,>>17<<]." ; 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