IRES-Mediated Protein Translation Overcomes Suppression by the p14ARF Tumor Suppressor Protein.

Internal ribosome entry sites (IRES elements) have attracted interest in cancer gene therapy because they can be used in the design of gene transfer vectors that provide bicistronic co-expression of two transgene products under the control of a single promoter. Unlike cellular translation of most mRNAs, a process that requires a post-translational 5' modification of the mRNA known as the cap structure, IRES-mediated translation is independent of the cap structure. The cellular conditions that may intervene to modulate IRES-mediated, cap-independent versus cap-dependent translation, however, remain poorly understood, although they could be critical to the choice of gene transfer vectors. Here we have compared the effects of the p14ARF (Alternate Reading Frame) tumor suppressor, a translational suppressor frequently overexpressed in cancer, on cap-dependent translation versus cap-independent translation from the EMCV viral IRES often used in bicistronic gene transfer vectors. We find that ectopic overexpression of p14ARF suppresses endogenous and ectopic cap-dependent protein translation, consistent with other studies. However, p14ARF has little or no effect on transgene translation initiated within an IRES element. This suggests that transgenes placed downstream of an IRES element will retain efficient translation of their gene products in the presence of high levels of ectopic or endogenous p14ARF, a finding that could be particularly relevant to therapeutic gene therapy strategies for cancer

Phosphorylation of CRN2 by CK2 regulates F-actin and Arp2/3 interaction and inhibits cell migration

CRN2 (synonyms: coronin 1C, coronin 3) functions in the re-organization of the actin network and is implicated in cellular processes like protrusion formation, secretion, migration and invasion. We demonstrate that CRN2 is a binding partner and substrate of protein kinase CK2, which phosphorylates CRN2 at S463 in its C-terminal coiled coil domain. Phosphomimetic S463D CRN2 loses the wild-type CRN2 ability to inhibit actin polymerization, to bundle F-actin, and to bind to the Arp2/3 complex. As a consequence, S463D mutant CRN2 changes the morphology of the F-actin network in the front of lamellipodia. Our data imply that CK2-dependent phosphorylation of CRN2 is involved in the modulation of the local morphology of complex actin structures and thereby inhibits cell migration

Specificity of pAb506P.

<p><b>(A)</b> Comparative titration curves of pAb506P on ELISA plates coated with a topo I peptide surrounding the serine 506 site, either in its phosphorylated or non-phosphorylated form. <b>(B)</b> Western analyses of H358 cell lysates (100 μg/lane) probed with pAb506P (lane 1) or with goat anti-topo I (lane 2). Arrows indicate positions of the 45 kDa species and full length topo I. <b>(C)</b> Topo I immunoprecipitation (goat anti-topo I C-terminus) followed by pAb506P Western of H358 cell lysates. Lane represents 200 μg starting material. <b>(D)</b>Western analysis of PS506 and actin in H358 cell lysates before (cntr) and after treatment with alkaline phosphatase (AP). <b>(E)</b> Western analysis of PS506 (using pAb506P), full length topo I (using goat anti-topo I) and tubulin in H358 cells before and after a 24 hr treatment of cells with 0.1 or 1 μM CPT.</p

Expression of PS506 in malignant, non-malignant, and benign tumor tissues.

<p><b>(A)</b> Representative PS506 Western blot of specimens of NSCLC and their paired non-malignant specimens (specimen pairs 8–13). H358 is the reference control for quantification of all PS506 blots. Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134929#pone.0134929.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134929#pone.0134929.t002" target="_blank">2</a> list the specimens analyzed and quantification of PS506 levels relative to H358. <b>(B)</b> Bar graph of the PS506 levels shown in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134929#pone.0134929.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134929#pone.0134929.t002" target="_blank">2</a>, grouped as paired malignant/non-malignant specimens and benign tumors. <b>(C)</b> Scatter plot of PS506 levels from Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134929#pone.0134929.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134929#pone.0134929.t002" target="_blank">2</a>, grouped as malignant (M), non-malignant (N), and benign (B) tumors. The p values were calculated by an unpaired t-test.</p

Correlation of CK2 activity, PS506 expression, and camptothecin sensitivity in cancer cell lines.

<p>(<b>A</b>) CK2 activity in cell lysates of the indicated cell lines. (<b>B</b>) Western analysis of PS506, total topo I, and actin in lysates of the indicated cell lines. Each lane contains 75 µg of cell lysate. Numbers below lanes refer to the intensities of the PS506 band relative to H358, as determined digitally. (<b>C</b>) Day 3 viability assays of the indicated cell lines following exposure to the indicated doses of camptothecin (CPT) for the first 18 h of the incubation.</p

Topo I cleavage complex formation and induction of DNA double-strand breaks.

<p>(A) Cleavage complex formation in untreated OVCAR-3 cells (bars 1,5), or 2 days after treatment with 20 moi Adp14 (bars 2,6), 10 nM of the CK2 activator 1-ethyl, 4,5 dicarbamoyl imidazole (bars 3,7), or both Adp14 and the CK2 activator (bars 4,8). Samples 5–8 were also treated with 10 µM of the ROS inducer pyocyanin. Cells were pulsed with [³H]-thymidine to label DNA, cleavage complexes were captured by K⁺SDS precipitation, and DNA was quantified by scintillation counting. (B) Western analysis of γ-H2A.X, an indicator of DNA double-strand break formation, in lysates of H358 cells subjected to the same treatments 1–8 as in part (A). Total H2A.X levels are shown as a control.</p

Chromatin association of topo I.

<p>(A) Rows 1–3: Topo I IP followed by Western analyses of total topo I, phosphoserine, and ARF was performed before (lane “C”) or 2 days after (lane “TBB”) treatment of H358 cells with TBB (10 µM for 1 h); rows 4 and 5: Western analyses of PS506 and total topo I in the same starting samples as in rows 1–3. Quantification of band densities indicated that TBB treatment reduced both P-ser and PS506 reactivity by ∼80%. (B) Histone H3 chromatin immunoprecipitation (ChIP) of untreated H358 cells (lane 1) or 2 days after treatment with 20 moi Adp14 (lane 2), TBB (10 µM, 1 h; lane 3), or both Adp14 and TBB (lane 4), followed by Western analyses of histone H3 and topo I. (C) The results of four independent ChIP analyses performed as in (B); bars represent the mean and standard deviation of chromatin-associated topo I levels in the treated cells relative to the untreated cells, quantified digitally from band intensities.</p