5 research outputs found

    PCTAIRE1-Knockdown Sensitizes Cancer Cells to TNF Family Cytokines

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    <div><p>While PCTAIRE1/PCTK1/Cdk16 is overexpressed in malignant cells and is crucial in tumorigenesis, its function in apoptosis remains unclear. Here we investigated the role of PCTAIRE1 in apoptosis, especially in the extrinsic cell death pathway. Gene-knockdown of <i>PCTAIRE1</i> sensitized prostate cancer PPC1 and Du145 cells, and breast cancer MDA-MB-468 cells to TNF-family cytokines, including TNF-related apoptosis-inducing ligand (TRAIL). Meanwhile, PCTAIRE1-knockdown did not sensitize non-malignant cells, including diploid fibroblasts IMR-90 and the immortalized prostate epithelial cell line 267B1. PCTAIRE1-knockdown did not up-regulate death receptor expression on the cell surface or affect caspase-8, FADD and FLIP expression levels. PCTAIRE1-knockdown did promote caspase-8 cleavage and RIPK1 degradation, while RIPK1 mRNA knockdown sensitized PPC1 cells to TNF-family cytokines. Furthermore, the kinase inhibitor SNS-032, which inhibits PCTAIRE1 kinase activity, sensitized PPC1 cells to TRAIL-induced apoptosis. Together these results suggest that PCTAIRE1 contributes to the resistance of cancer cell lines to apoptosis induced by TNF-family cytokines, which implies that PCTAIRE1 inhibitors could have synergistic effects with TNF-family cytokines for cytodestruction of cancer cells.</p></div

    Targeting PCTAIRE1 using an inducible shRNA vector in PPC1 cells.

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    <p>(A) PPC1 cells stably containing inducible shRNAs targeting different sites on PCTAIRE1 mRNA (shRNA#1, #2) or scramble-control were cultured for 48 hours with doxycycline (Dox, 100 ng/ml). Protein lysates were generated, normalized for total protein concentration, and analyzed by SDS-PAGE/immunoblotting using antibodies for PCTAIRE1 (top) and beta-actin (bottom). (B, C) PPC1 cells were cultured with (ON) or without (OFF) 100 ng/ml Dox for 48 hours, then stimulated with various concentrations of either anti-Fas antibody CH-11 (B) or TRAIL (C). After 24 hours, cellular ATP levels were measured, and the data expressed as a ratio relative to cells cultured without anti-Fas or TRAIL (mean ± SD; n = 3). (D, E) Clonogenic survival assays. PPC1 cells stably containing inducible shRNAs (scramble or shRNA#2) were cultured with (ON) or without (OFF) 100 ng/ml Dox for 48 hours, then stimulated with anti-Fas antibody (CH-11, 10 ng/ml) or TRAIL (20 ng/ml). Cells were cultured for 3 days before fixing and staining with 0.5% crystal violet dye.</p

    PCTAIRE1 knockdown sensitizes PPC1 cells to anti-Fas antibody and TRAIL.

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    <p>(A) PPC1 cells were transfected with scrambled RNA or three different siRNAs targeting PCTAIRE1 (siRNAs 1472, 1566, 1656). Lysates from cells at 48 hours post-siRNA transfection were prepared, normalized for total protein content, and aliquots were analyzed by immunoblotting using anti-PCTAIRE1 (top) or anti-alpha-tubulin (bottom) antibodies. (B, C) PPC1 cells were transfected with control RNA (purple “x”) or various siRNAs targeting PCTAIRE1 (blue diamonds, 1472; red squares, 1566, green triangles, 1656). After 48 hours, cells were stimulated with either anti-Fas antibody (CH-11) (B) or TRAIL (C) at various concentrations as indicated. After 24 hours, cellular ATP levels were measured as a surrogate indicator of cell viability using Cell Titer Glo reagents, and the data are expressed as the ratio between cells cultured with and without anti-Fas (B), and TRAIL (C). (D) Apoptosis analyses were performed using an annexinV kit. PPC1 cells were transfected with scramble RNA of siRNAs targeting PCTAIRE1. After 48 hours, cells were treated with anti-Fas antibody (100 ng/ml) or TRAIL (50 ng/ml) for 4 hours. *P < 0.05, ***P < 0.001 by t-test. All data represent mean ± SD (n = 3). (E, F) Clonogenic survival assays. PPC1 cells were seeded in 6 well (35 mm) dishes at 1.0 x 10<sup>5</sup> cells per well and then reverse-transfected with scramble-control or PCTAIRE1-targeting siRNAs as indicated. After 48 hours, anti-Fas antibody (E, 10 ng/ml) or TRAIL (F, 20 ng/ml) was added and cells were cultured for 3 days before fixing and staining with 0.5% crystal violet dye. (G, H) PPC1 cells were transfected with control RNA or three different siRNAs targeting PCTAIRE1. After 48 hours, the cells were stimulated with Fas (G, 10 ng/ml) or TRAIL (H, 10 ng/ml). After 3 hours, cell lysates were prepared, normalized for total protein content, and analyzed by immunoblotting using an antibody specific for cleaved caspase-8. The partially cleaved 43/41 kDa bands and fully cleaved 18 kDa band are indicated, as are molecular weight markers (kDa). The blot was reprobed with beta-actin antibody as a loading control. (I) PPC1 cells were transfected with scramble-control or siRNA targeting PCTAIRE1 (si-1472). After 48 hours, cells were or were not stimulated with anti-Fas antibody (CH-11, 10 ng/ml). After 3 hours, cell lysates were harvested and immunoprecipitated with anti-caspase 8 antibody, followed by immunoblotting with the indicated antibodies. The red arrowheads indicate non-specific bands.</p

    Transformed cells are preferentially sensitive to PCTAIRE1 knockdown-effect with TRAIL treatment.

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    <p>(A-C) Human diploid fibroblasts IMR-90 (A), 267B1 immortalized normal prostate epithelial cells (B), and K-ras transformed 267B1 (267B1/K-ras) cells (C) were transfected with scrambled RNA or three different siRNAs targeting PCTAIRE1 (siRNAs: si-1472, si-1566, si-1656). Lysates of cells 72 hours post-transfection were prepared and analyzed by immunoblotting using rabbit anti-PCTAIRE1 (top) or anti-beta-actin (bottom) antibodies. (D-F) IMR-90, 267B1 and 267B1/K-ras cells were transfected with siRNAs. After 48 hours, cells were stimulated with TRAIL at various concentrations as indicated. After 24 hours, cellular ATP levels were measured, and the data expressed as a ratio between cells cultured with and without TRAIL (mean ± SD; n = 3).</p

    PCTAIRE1-knockdown modulates RIPK1 expression.

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    <p>(A) PPC1 cells were transfected with control RNA or three different siRNAs targeting PCTAIRE1. After 48 hours, cell lysates were prepared with RIPA buffer, normalized for total protein content, and analyzed by immunoblotting using the indicated antibodies. (B) PPC1 cells were transfected with indicated siRNAs for 48 hours, whereupon cell lysates were collected in 1x Laemmli buffer and subjected to immunoblotting analysis for RIPK1 (top) and beta-actin (middle). (C, D) PPC1 cells were transfected with the indicated siRNAs. After 48 hours, cells were treated with the proteasome inhibitor MG-132 (10 μM) for 5 hours before preparation of cell lysates (C: 1x Laemmli buffer, D: RIPA buffer). (E) PPC1 cells were transfected with control RNA or siRNA targeting RIPK1. After 48 hours, cell lysates were prepared in RIPA buffer, normalized for total protein content, and analyzed by immunoblotting using the indicated antibodies. (F, G) PPC1 cells were transfected with control RNA or siRNA targeting RIPK1. After 48 hours, cells were stimulated with either anti-Fas antibody (CH-11) (F) or TRAIL (G) at various concentrations as indicated. After 24 hours, cellular ATP levels were measured as a surrogate indicator of cell viability using Cell Titer Glo reagents, and the data expressed as a ratio between cells cultured with and without anti-Fas (F) or TRAIL (G). (H) Clonogenic survival assays. PPC1 cells were seeded in 6 well (35 mm) dishes at 1.0 x 10<sup>5</sup> cells per well, and then reverse-transfected with scramble-control, PCTAIRE1-targeting siRNA (si-1472), or RIPK1-targeting siRNA as indicated. After 48 hours, anti-Fas antibody (top, 2.5 ng/ml) or TRAIL (bottom, 10 ng/ml) was added and cells were cultured for 3 days before fixing and staining with 0.5% crystal violet dye.</p
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