2002;4(2):75C85. for the treatment of medulloblastoma. Daoy cells were transfected with control, Mnk1, Mnk2 and Mnk1+Mnk2 siRNAs. After 48 hours, cells were treated with rapamycin (20 nM) for 90 min, as indicated. AKOS B018304 Cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against the phosphorylated form of eIF4E (pSer-209). The same membrane was stripped and reprobed with an antibody for eIF4E. mRNA expression of Mnk1 and Mnk2 genes from cells transfected with the indicated siRNAs from your same experiment shown on the panel, was assessed by quantitative RT-PCR in triplicates, using GAPDH for normalization. Data are expressed as percentages of control siRNA transfected cells. (C) Mnk1/2+/+, Mnk1-/-, Mnk2-/- and Mnk1/2-/- AKOS B018304 (DKO) MEFs were treated with rapamycin (20 nM) for 90 min. Equivalent amounts of protein were resolved by SDS-PAGE and immunoblotted with antibodies against phosphorylated eIF4E (pSer-209) or p70-S6K (pThr-389). Membranes were stripped and reprobed with antibodies for eIF4E, p70-S6K and GAPDH. There has been previous evidence that MAPKs activate Mnk1 for inducible phosphorylation of eIF4E, whereas Mnk2 mainly contributes to eIF4E’s basal, constitutive phosphorylation [31]. To define whether rapamycin-induced increase in eIF4E phosphorylation is usually mediated by Mnk1 or Mnk2, we knocked down Mnk1 or Mnk2 in Daoy medulloblastoma cells, and examined the effects of such knockdown on rapamycin-inducible eIF4E phosphorylation. Rapamycin treatment resulted in an increase in eIF4E phosphorylation in cells in which Mnk1 was knocked down, but not in cells with selective Mnk2 knockdown (Fig. ?(Fig.4B).4B). These findings suggested that during treatment of medulloblastoma cells with rapamycin there is selective activation of Mnk2, but not Mnk1, for phosphorylation of eIF4E. Comparable results were observed in Mnk knockout MEFs [31, 32], where rapamycin increased eIF4E phosphorylation Mouse monoclonal to CD22.K22 reacts with CD22, a 140 kDa B-cell specific molecule, expressed in the cytoplasm of all B lymphocytes and on the cell surface of only mature B cells. CD22 antigen is present in the most B-cell leukemias and lymphomas but not T-cell leukemias. In contrast with CD10, CD19 and CD20 antigen, CD22 antigen is still present on lymphoplasmacytoid cells but is dininished on the fully mature plasma cells. CD22 is an adhesion molecule and plays a role in B cell activation as a signaling molecule in Mnk1-/- MEFs, but failed to do so in Mnk2-/- or Mnk1/2-/- MEFs (Fig. ?(Fig.4C4C). In subsequent studies, we sought to determine whether combined treatment of medulloblastoma cells with Mnk and mTOR inhibitors results in enhanced antineoplastic effects. Daoy cells were treated with the Mnk inhibitor “type”:”entrez-protein”,”attrs”:”text”:”CGP57380″,”term_id”:”877393391″,”term_text”:”CGP57380″CGP57380 and either rapamycin or OSI-027, and cells were subjected to cell viability assays. Increasing concentrations of “type”:”entrez-protein”,”attrs”:”text”:”CGP57380″,”term_id”:”877393391″,”term_text”:”CGP57380″CGP57380 alone only marginally inhibited cell proliferation in these cells (Fig. ?(Fig.5A).5A). However, when “type”:”entrez-protein”,”attrs”:”text”:”CGP57380″,”term_id”:”877393391″,”term_text”:”CGP57380″CGP57380 was combined with increasing concentrations of rapamycin, it enhanced rapamycin’s antiproliferative effect in a dose-dependent manner (Fig. ?(Fig.5A,5A, upper panel). By contrast, “type”:”entrez-protein”,”attrs”:”text”:”CGP57380″,”term_id”:”877393391″,”term_text”:”CGP57380″CGP57380 failed to enhance the antiproliferative effects of increasing concentrations of OSI-027 (Fig. ?(Fig.5A,5A, lesser panel). Comparable results were obtained when cell counts were used (Fig. ?(Fig.5B).5B). Taken together, our results suggest that selective mTORC1 inhibition in medulloblastoma cells results in engagement of a Mnk2-dependent survival mechanism that can be counteracted by concomitant Mnk inhibition. In studies in which the effects of combination therapies on anchorage-independent growth of Daoy medulloblastoma cells were assessed, we found enhanced effects by the combinations of mTOR and Mnk inhibitors AKOS B018304 (Fig. ?(Fig.5C).5C). Knockdown of Mnk2, but not Mnk1, using specific siRNAs enhanced rapamycin-dependent inhibition of anchorage-independent growth, as compared to rapamycin alone. (Fig. ?(Fig.5D5D). Open in a separate window Physique 5 Simultaneous Mnk inhibition increases rapamycin-mediated inhibition of cell proliferation and colony formation(A) Daoy cells were incubated for five days with increasing concentrations of “type”:”entrez-protein”,”attrs”:”text”:”CGP57380″,”term_id”:”877393391″,”term_text”:”CGP57380″CGP57380 (1, 5, 10, 50 M) in the presence or absence of increasing concentrations of rapamycin (1, 5, 10, 50 nM, upper panel) or OSI-027 (1, 5, 10, 50 M, lower panel). Subsequently, cells were subjected to.

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