This Article Abstract Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Chen, S. Articles by Wang, C.-Y. Search for Related Content PubMed PubMed Citation Social Bookmarking                   What's this?

Potentiation of Tumor Necrosis Factor-mediated Apoptosis of Oral Squamous Cell Carcinoma Cells by Adenovirus-mediated Gene Transfer of NF-B Inhibitor

¹ Laboratory of Molecular Signaling and Apoptosis, Department of Biologic and Materials Science,
² Program in Oral Health Science, School of Dentistry, and
³ Program in Cellular and Molecular Biology, University of Michigan, 1011 N. University Ave., Ann Arbor, MI 48109-1078;

View larger version (72K): [in this window] [in a new window]   Figure 1. Expression of SR-IB by adenovirus-mediated gene transfer inhibits phosphorylation of IB induced by TNF. (A) Expression of SR-IB after Adv-SR-IB transduction. SCC15 cells were infected with adenoviruses expressing SR-IB or LacZ at a MOI of 200 for 4 hrs. Twenty-four hrs after infection, cells were treated with TNF (10 ng/mL) for the indicated time points. The whole-cell proteins were extracted and probed with polyclonal antibodies against IB. (B) SR-IB inhibited phosphorylation of IB induced by TNF. Both viral infection and cell treatment were performed as described in (A). Whole-cell extracts were probed with polyclonal antibodies against phospho-specific IB. As an internal control, the blots were stripped and re-probed with monoclonal antibodies against -tubulin.

View larger version (168K): [in this window] [in a new window]   Figure 2. Adv-SR-IB sensitizes SCC 15 cells to TNF killing. (A) Photograph of cells after TNF treatment. Cells were infected with Adv-SR-IB or Adv-LacZ as described in Fig. 1A. One day later, cells were treated with TNF (10 ng/mL) for 24 hrs and photographed under phase-contrast microscopy. (B) SR-IB sensitized cells to TNF killing. After TNF treatment, cells were harvested and incubated with 0.1% trypan blue. Dead and living cells were counted separately. Assays were performed in triplicate, and the results represent average values from three independent experiments. Statistical differences between groups were determined by Student's t test. Error bars represent standard deviation. *p < 0.01. (C) SR-IB enhances TNF-induced DNA fragmentation. Supernatants from (B) were collected and measured with a cell death ELISA kit. Assays were performed in duplicate, and the results represent average values from three independent experiments. Statistical differences between groups were determined by Student's t test. Error bars indicate standard deviation. *p < 0.01.

View larger version (12K): [in this window] [in a new window]   Figure 3. Adv-SR-IB renders oral SCC5 cells sensitive to TNF killing. Both viral infection and TNF treatment were performed as described in Fig. 2. Cells were harvested and incubated with 0.1% trypan blue. Dead and living cells were counted separately. Assays were performed in duplicate, and the results represent average values from three independent experiments. Error bars represent standard deviation. *p < 0.01.

View larger version (33K): [in this window] [in a new window]   Figure 4. TNF activates the caspase cascade in SCC cells after Adv-SR-IB transduction. (A) TNF induces caspase-8 activity after Adv-SR-IB transduction. Viral infection was performed as described in Fig. 1A. After infection, cells were treated with TNF (10 ng/mL) for the indicated time points. Whole-cell extracts were prepared and incubated with the specific caspase-8 substrate pNA-IETD (100 µM) at 37°C for 8 hrs. The reaction was measured with a plate reader at 405 nm. Assays were performed in triplicate, and the results represent average values from two independent experiments. Statistical differences between groups were determined by Student's t test. Error bars represent standard deviation. *p < 0.01. (B) TNF induces caspase-3 activity after Adv-SR-IB transduction. Whole-cell extracts were incubated with the specific caspase-3 substrate pNA-DEVD (100 µM) at 37°C for 16 hrs. Error bars represent standard deviation. *p < 0.01.

CiteULike

Connotea

Del.icio.us

Digg

Reddit

Technorati