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First published online 29 August 2006
doi: 10.1242/jcs.03102

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Adenoviral vector saturates Akt pro-survival signaling and blocks insulin-mediated rescue of tumor-necrosis-factor-induced apoptosis

¹ Biotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
² Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
³ Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
⁴ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

View larger version (27K): [in a new window]   Fig. 1. Adv infection sensitizes human epithelial cells to TNF-mediated apoptosis. (A) HT-29 cells were infected with either Adv.ß-gal or Adv.empty (1.4x1010 v.p./ml) or treated with buffer only and then stimulated with either 100 ng/ml TNF or carrier only. Cells were collected 48 hours after TNF addition, stained for either caspase-cleaved cytokeratin and cleaved (active) caspase 3 (white bars) or annexin V/PI (gray bars), and analyzed by flow cytometry. Adv.ß-gal was used for all subsequent experiments. (B) Sample flow cytometry plots for anti-caspase-cleaved cytokeratin and anti-cleaved (active) caspase 3 in Adv-sensitized and control cells treated with TNF. (C) HT-29 cells were infected with two concentrations of Adv (0.7x1010 and 1.4x1010 v.p./ml) for 0, 1, 3, 6 and 12 hours. The total number of Adv copies per cell was quantified by PCR. Values represent mean total ß-gal DNA normalized to mean total ß-actin DNA for duplicate samples. Variance in the Adv copy values ranged from 10 to 30% of the mean value. In parallel, HT-29 cells subject to the same infection conditions were treated with 100 ng/ml TNF and collected after 48 hours. Cleaved caspase 3-cytokeratin measurements are plotted as the mean apoptosis (above control level) of three biological replicates. (D) HeLa cells and HT-29 cells were infected with increasing doses of Adv (expressed as multiplicities of infection, p.f.u./cell) and stimulated with TNF or carrier, similar to panel A. Cells were collected 24 hours (HeLa) or 48 hours (HT-29) post-TNF treatment. Cleaved caspase 3-cytokeratin measurements are plotted as the mean of three biological replicates ± s.e.m.

View larger version (54K): [in a new window]   Fig. 2. Adv infection upregulates TNF-induced pro- and anti-apoptotic signaling. (A) The signaling pathways induced by TNF (blue) and insulin (green). The kinases measured (MK2, IKK, JNK1, Akt and ERK) are highlighted in yellow. Figure is modified and published with permission from the American Society for Biochemistry and Molecular Biology (Gaudet et al., 2005). (B-D) Dynamic activation status and inhibitor response data for the MK2 pathway (B), IKK pathway (C) and Akt pathway (D) in uninfected cells treated with 100 ng/ml TNF (TNF; blue) and Adv-infected cells treated with 100 ng/ml TNF (Adv + TNF; red). Lysates were collected at 0, 5, 15, 30, 60 and 90 minutes and 2, 4, 8, 12, 16, 20 and 24 hours, and kinase activity was measured by a high throughput kinase activity assay and normalized to total protein content. Results are plotted as the mean relative activation of two biological replicates normalized to activity of the untreated control at 0 minutes (i.e. uninfected cells). The significance of the difference between each pair of curves was calculated by two-way ANOVA (MK2: P<0.01; IKK: P<0.001; Akt: P<10-4). Apoptosis in the presence of kinase inhibition was measured by flow cytometry for cleaved caspase-cytokeratin. The following inhibitor concentrations were used: 10 µM SB202190 (SB); 20 µM SC-514 (SC); 20 µM LY294002 (LY). Cells were collected at 24 hours, rather than 48 hours, to minimize baseline apoptosis resulting from inhibition. Measurements are plotted as the mean percentage change in apoptosis resulting from inhibition (i.e. mean percentage apoptosis in the presence of inhibitor - mean percentage apoptosis without inhibitor) of three (SC) or six (SB, LY) biological replicates ± s.e.m. Note that LY plot ranges from -40 to +40 (compared with -20 to +20 for other inhibitor plots). Changes are labeled as significant (*) if P<0.05. (E) Adv-TNF synergy via pro-apoptotic p38-MK2 signaling and anti-apoptotic PI3K-Akt signaling. Synergy is illustrated schematically as an operational amplifier, with the strength of the Adv or TNF input into either the MK2 or Akt signaling time-course indicated by line weight. Amplification of the inputs results in activation (MK2) or inhibition (Akt) of apoptosis.

View larger version (34K): [in a new window]   Fig. 3. Insulin increases Akt activity but does not reverse TNF-induced apoptosis or reduce MK2 activity in Adv-infected cells. Adv- and IFN-sensitized HT-29 cells were treated with TNF (100 ng/ml and 50 ng/ml, respectively) in the presence and absence of (A-C) 100 nM insulin or (D-F) 20 µM LY294002. Cells were lysed at 12 hours and assayed for kinase activity, or collected after 24 hours and assayed for apoptosis by flow cytometry for cleaved caspase-cytokeratin. (A,D) Change in TNF-induced cell death. Measurements are plotted as the mean of six biological replicates (two independent experiments) ± s.e.m. (B,E) change in TNF-induced Akt activity. Measurements are plotted as the mean of six biological replicates (two independent experiments) ± s.e.m. (C,F) Change in TNF-induced MK2 activity in Adv-sensitized cells. Measurements are plotted as the mean of three biological replicates ± s.e.m. Changes are labeled as significant (*) if P<0.05.

View larger version (37K): [in a new window]   Fig. 4. Adv infection saturates phosphorylation of downstream Akt effectors. (A) Cells were treated as described in Fig. 3, lysed at 12 hours and analyzed by western blot by probing with anti-pGSK-3/ß and anti-Akt-pSub. Experimental conditions are the same as those described in Fig. 3. 10% fetal bovine serum (FBS) stimulation for 15 minutes was used as the positive-control stimulus. 50 µg protein was loaded into each lane. (B,C) Adv-sensitized and IFN-sensitized (B) pGSK-3 and (C) Akt-pSub42kDa bands were quantified by densitometry and normalized to the sensitized (Adv or IFN) 0-min band. Measurements are plotted as the mean of three biological replicates ± s.e.m. Changes are labeled as significant (*) if P<0.05.

View larger version (13K): [in a new window]   Fig. 5. Adv-infected are trapped near the plateau of a global Akt-survival dose-response curve. (A,B) Following Adv or IFN sensitization, cells were treated with TNF (100 ng/ml and 50 ng/ml, respectively), TNF + LY294002 (20 µM), TNF + insulin (100 nM), or TNF + LY294002 + insulin (B only). Conditions were chosen to generate a range of Akt activities. Measurements were taken at 12 hours for Akt activity and effector phosphorylation and at 24 hours for apoptosis as described in Fig. 3. (A) Cell survival versus fold activation of Akt activity. Akt activities were normalized to sensitized 0-min values from A. Akt measurements are the mean of six biological replicates. Survival measurements are 100% - the mean of six biological replicates. (B) Plot of survival (from A) versus effector phosphorylation levels quantified in Fig. 4. Solid red markers indicate IFN-sensitized conditions and open red markers indicate Adv-sensitized conditions (same as in A). (A,B) Sigmoid function and parameters were calculated as described in Materials and Methods. Transition steepness (Ts) and the upper and lower bounds of the 90% confidence interval (parentheses) are labeled on the graphs.

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