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Home » Ohno S, Naito Y, Mukai S, Yabuta N, Nojima H

Ohno S, Naito Y, Mukai S, Yabuta N, Nojima H

Ohno S, Naito Y, Mukai S, Yabuta N, Nojima H. assay, in the presence of camptothecin, an inducer of DNA double-strand breaks. Moreover, nude mice harboring Myc-ELAS1-expressing SAS cells lived longer than mice harboring Myc-vector-expressing SAS cells, suggesting the usefulness of ELAS1 mutations [16C19]. Among common cancers, we selected prostate cancer and tongue cancer cell lines to further study ELAS1 function. DU145 cells harboring the P223L and V274F point mutations but with the wild-type (WT) p53-S46 residue [20] are less sensitive to docetaxel than LNCaP and C4-2 cells, which express functional p53 [21]. Because this phenomenon is due to increased p53-S15 phosphorylation [21], it MM-102 TFA remains undetermined if ELAS1-mediated apoptosis also occurs in DU145 cells through increased p53-S46 phosphorylation. As a tongue cancer cell line, SAS appears to be suitable to examine the apoptotic function of ELAS1 because it harbors the WT p53-S46 residue, although it has an E336X (X means a stop codon) mutation, generating a truncated p53 protein, according to the mutation list in the TP53 website ( A large number of mutations listed in this website would play a role in personalized medicine by providing targets for drug development and new therapeutic approaches [22]. The aim of this study was to show that ELAS1 is useful as an adjuvant that helps to kill cancer cells with much lower doses of IR, CPT, and irinotecan. To this end, we examined DU145 and SAS cells. Moreover, to develop an efficient method to deliver the ELAS1 peptide into cancer cells, we prepared a recombinant adenovirus that expressed both ELAS1 and WT p53 protein and found that it efficiently killed p53-deficient SAS cells. We also found that Mouse monoclonal antibody to CaMKIV. The product of this gene belongs to the serine/threonine protein kinase family, and to the Ca(2+)/calmodulin-dependent protein kinase subfamily. This enzyme is a multifunctionalserine/threonine protein kinase with limited tissue distribution, that has been implicated intranscriptional regulation in lymphocytes, neurons and male germ cells ELAS1 could be shortened from 29 aa to ca. 10 aa without loss of its apoptosis-inducing function. These results demonstrate the general usefulness of ELAS1 for use at the bedside in the future. RESULTS ELAS1 causes apoptosis in DU145 cancer cells We previously showed that the ELAS1 peptide efficiently causes apoptosis in human osteosarcoma U2OS cells through inhibition of the CycG1-B association, leading to stabilization and activation of p53 [9]. We investigated if this phenotype is applicable to other more prevalent cancers. We first tested human prostate cancer by generating human adenocarcinoma DU145 cells that expressed doxycycline (Dox)-inducible Myc-vector or Myc-ELAS1. Western blot (Wb) analysis confirmed the successful construction of these DU145/Tet-On cells expressing Myc-vector or Myc-ELAS1 in a Dox-dependent manner (Figure ?(Figure1A).1A). Indeed, Myc-ELAS1 (green arrowhead) migrated slower than Myc-vector alone (purple arrow). Flow cytometry (FC) revealed that Dox-dependent expression of Myc-vector alone and Myc-ELAS1 had no effect on cell cycle progression (column non-treated (NT) in Figure ?Figure1B).1B). The subG1 population of Myc-ELAS1-expressing DU145 cells increased to 10.69% and 21.18% at 48 h after exposure to 1 and 10 Gy -IR, respectively (red arrows in Figure ?Figure1B).1B). By contrast, no change was observed in DU145 cells expressing Myc-vector alone (blue arrows in Figure ?Figure1B).1B). Bar graphs of the data clearly show the induction of apoptosis by Myc-ELAS1 (red arrows in Figure ?Figure1C)1C) compared with Myc-vector alone (blue arrows in Figure ?Figure1C).1C). Wb confirmed that Myc-ELAS1-expressing DU145 cells showed a band corresponding to p53-pS46 (red arrowhead in Figure ?Figure1D)1D) at 48 h after treatment with 1 Gy (lane 8) or 10 Gy (lane 10) -IR, even when the p53 protein level was not largely increased or rather decreased (black arrowhead in Figure ?Figure1D).1D). To examine if the increased subG1 population was actually derived from apoptotic cell death, we conducted the TUNEL assay. Indeed, apoptosis of Myc-ELAS1-expressing DU145 cells was increased at 24 and 48 h after treatment with 1 Gy or 10 Gy -IR (Supplementary Figure 1). These MM-102 TFA results suggest that point mutations (P223L and V274F) of p53 protein do not hamper the ELAS1-mediated apoptosis through phosphorylation of p53-pS46. Open in a separate window Figure 1 Exogenous expression of ELAS1 causes apoptotic death of DU145 cells after dsDNA insults(A) Wb was conducted to show the successful establishment of MM-102 TFA DU145/Tet-On cells expressing Myc-vector or Myc-ELAS1 in the absence (-) or presence (+) of Dox. The purple arrow and green arrowhead indicate Dox-inducible bands for Myc protein and Myc-ELAS1 protein, respectively. (B) FC analysis. DU145/Tet-On cells stably expressing Myc-vector alone or Myc-ELAS1 were treated with 1 or 10 Gy -IR for the indicated duration (h) in the presence of Dox. Cells were stained with propidium iodide (PI) and the cell cycle profiles were determined by FC. The percentages correspond to the sub-G1 population of cells. (C) The bar graphs show the percentages of.


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