Furthermore, targeted deletion of a major PI-3K antagonist, PTEN, results in safety of T cells from SEB-induced deletion up-regulation (data not shown). within cells. Because levels of Bcl-2 within cells are crucial to antiapoptotic activity, reducing Bcl-2 could be a mechanism to sensitize cells to apoptosis. By detoxifying ROS, antioxidants may consequently reverse the ROS-induced decrease in Bcl-2 and prevent apoptosis. Several studies suggest a reciprocal relationship between ROS and Bcl-2 levels within cells (6C8). Therefore, in divergent cell types, decreases in ROS correlate with raises in Bcl-2 levels and vice versa. Recently we showed that, down-regulation within triggered T cells. Taken together, these results suggest a two-signal model for triggered T cell apoptosis. The first signal is driven by ROS down-regulation of Bcl-2, which is necessary, but not adequate, to total apoptosis. The second signal requires the manifestation of Bim. Methods T Cell Purification for Microarray Analysis. T cells were triggered in C57BL/10 (= 16) mice by i.v. injection of 150 g of staphylococcal enterotoxin B (SEB; Toxin Technology, Sarasota, FL). Mice were killed 48 h later on, and lymph nodes cells were cultured with or without 150 M MnTBAP for 7 h at 37C and then stained with fluorescently labeled antibodies to CD4, CD8, V8, and I-Ab (Pharmingen). CD4+ CD8+ V8+ I-Ab- cells were sorted by using a high-speed MoFlo circulation cytometer (Cytomation, Fort Collins, CO). RNA extraction, cDNA synthesis, and gene microarray analysis were performed as explained (11, 12) Mice. C57BL/10 and C57BL/6J (BL/6) mice were purchased from your Jackson Laboratory. (Pharmingen), then washed, permeabilized with 0.03% saponin (Sigma), stained with anti-Bcl-2 or anti-Bcl-xL antibodies (Pharmingen), washed, and stained with secondary antibodies. Data were collected on a FACSCalibur flow cytometer and analyzed with CELLQUEST software (Becton Dickinson). T cell death was assessed by staining with anti-V8.x-FITC and propidium iodide (0.5 g/ml) as described (10). Percent inhibition of cell death was calculated as follows: [(percent dead without MnTBAP – percent dead with MnTBAP)/percent dead without MnTBAP] 100. Superoxide levels were determined by using dihydroethidium as described (10). Production and Use of Retroviruses. pMSCV-IRES-GFP (MiG, a plasmid derived from the murine stem cell virus containing an internal ribosome entry sequence followed by a GFP cassette) was a kind gift from William Sha (University of California, Berkeley), and pCLEco (a plasmid encoding cDNAs) was a kind gift from Inder Verma (The Salk Institute, La Jolla, CA) (14). pMSCV-IRES-Thy1.1 (MiT) was derived from MiG as described (12). Mouse cDNAs encoding catalase, Mn superoxide dismutase (MnSOD), and Bcl-2 were cloned by means of PCR amplification from activated T cell cDNA, and inserts were confirmed by restriction digestion and DNA sequencing. MnSOD Q143A was generated from MiT MnSOD by site-directed mutagenesis. Plasmid DNA was transformed and amplified in DH5 and purified by using the QIAfilter Mega kit (Qiagen, Valencia, CA). Retroviruses were produced by cotransfection of 293 human embryonic kidney cells with pCLEco and the MiT plasmid of interest by using calcium phosphate as described (9, 12). After transduction, cells were stained with various fluorescently labeled antibodies, all of which were supplied by Pharmingen, and analyzed by flow cytometry. Live and dead cells were distinguished by their forward/side scatter properties as described (12, 15). Results Properties of Cells Used for Gene Array Analysis. V8+ T cells were activated by injection of SEB into C57BL/10 mice, isolated 2 days later, cultured for 7 h, and purified from MnTBAP-treated or control-treated cultures by using high-speed cell sorting. After sorting, both treated and control T cells were roughly 98% V8+ (Table 1). For both groups, 0.18% of the sorted cells were.pMSCV-IRES-Thy1.1 (MiT) was derived from MiG as described (12). act to down-regulate endogenous Bcl-2 levels within cells. Because levels of Bcl-2 within cells are critical to antiapoptotic activity, decreasing Bcl-2 could be a mechanism to sensitize cells to apoptosis. By detoxifying ROS, antioxidants may therefore reverse the ROS-induced decline in Bcl-2 and prevent apoptosis. Several studies suggest a reciprocal relationship between ROS Rabbit polyclonal to COFILIN.Cofilin is ubiquitously expressed in eukaryotic cells where it binds to Actin, thereby regulatingthe rapid cycling of Actin assembly and disassembly, essential for cellular viability. Cofilin 1, alsoknown as Cofilin, non-muscle isoform, is a low molecular weight protein that binds to filamentousF-Actin by bridging two longitudinally-associated Actin subunits, changing the F-Actin filamenttwist. This process is allowed by the dephosphorylation of Cofilin Ser 3 by factors like opsonizedzymosan. Cofilin 2, also known as Cofilin, muscle isoform, exists as two alternatively splicedisoforms. One isoform is known as CFL2a and is expressed in heart and skeletal muscle. The otherisoform is known as CFL2b and is expressed ubiquitously and Bcl-2 levels within cells (6C8). Thus, in divergent cell types, decreases in ROS correlate with increases in Bcl-2 levels Doxercalciferol and vice versa. Recently we showed that, down-regulation within activated T cells. Taken together, these results suggest a two-signal model for activated T cell apoptosis. The first signal is driven by ROS down-regulation of Bcl-2, which is necessary, but not sufficient, to complete apoptosis. The second signal requires the expression of Bim. Methods T Cell Purification for Microarray Analysis. T cells were activated in C57BL/10 (= 16) mice by i.v. injection of 150 g of staphylococcal enterotoxin B (SEB; Toxin Technology, Sarasota, FL). Mice were killed 48 h later, and lymph nodes cells were cultured with or without 150 M MnTBAP for 7 h at 37C and then stained with fluorescently labeled antibodies to CD4, CD8, V8, and I-Ab (Pharmingen). CD4+ CD8+ V8+ I-Ab- cells were sorted by using a high-speed MoFlo flow cytometer (Cytomation, Fort Collins, CO). RNA extraction, cDNA synthesis, and gene microarray analysis were performed as described (11, 12) Mice. C57BL/10 and C57BL/6J (BL/6) Doxercalciferol mice were purchased from The Jackson Laboratory. (Pharmingen), then washed, permeabilized with 0.03% saponin (Sigma), stained with anti-Bcl-2 or anti-Bcl-xL antibodies (Pharmingen), washed, and stained with secondary antibodies. Data were collected on a FACSCalibur flow cytometer and analyzed with CELLQUEST software (Becton Dickinson). T cell death was assessed by staining with anti-V8.x-FITC and propidium iodide (0.5 g/ml) as described (10). Percent inhibition of cell death was calculated as follows: [(percent dead without MnTBAP – percent dead with MnTBAP)/percent dead without MnTBAP] 100. Superoxide levels were determined by using dihydroethidium as described (10). Production and Use of Retroviruses. pMSCV-IRES-GFP (MiG, a plasmid derived from the murine stem cell virus containing an internal ribosome entry sequence followed by a GFP cassette) was a kind gift from William Sha (University of California, Berkeley), and pCLEco (a Doxercalciferol plasmid encoding cDNAs) was a kind gift from Inder Verma (The Salk Institute, La Jolla, CA) (14). pMSCV-IRES-Thy1.1 (MiT) was derived from MiG as described (12). Mouse cDNAs encoding catalase, Mn superoxide dismutase (MnSOD), and Bcl-2 were cloned by means of PCR amplification from activated T cell cDNA, and inserts were confirmed by restriction digestion and DNA sequencing. MnSOD Q143A was generated from MiT MnSOD by site-directed mutagenesis. Plasmid DNA was transformed and amplified in DH5 and purified by using the QIAfilter Mega kit (Qiagen, Valencia, CA). Retroviruses were produced by cotransfection of 293 human embryonic kidney cells with pCLEco and the MiT plasmid of interest by using calcium phosphate as described (9, 12). After transduction, cells were stained with various fluorescently labeled antibodies, all of which were supplied by Pharmingen, and analyzed by flow cytometry. Live and dead cells were distinguished by their forward/side scatter properties as described (12, 15). Results Properties of Cells Used for Gene Array Analysis. V8+ T cells were activated by injection of SEB into C57BL/10 mice, isolated 2 days later, cultured for 7 h, and purified from MnTBAP-treated or control-treated cultures by using high-speed cell sorting. After sorting, both treated and control T cells were roughly 98% V8+ (Table 1). For both groups, 0.18% of the sorted cells were I-Ab+ and the remaining non-V8+ cells were mostly resting T cells expressing a V other than V8. To determine whether MnTBAP had the desired effect on the T cells in this experiment, samples from the two groups of T cells were cultured for an additional 5 h (total of 12 h) and assessed for cell death. Indeed, culture with MnTBAP significantly decreased activated T cell death (Table 1; 0.00001, two-sample test; MINITAB for Windows). Table 1. Properties of sorted T cells used for Affymetrix GeneChip analysis Activated T cells cultured V8, % I-Ab, % V8+ cells that died,* % Transcript levels for GAPDH Control 97.75 0.47 0.18 0.11 59.35 3.82 16,285 2,551 MnTBAP 98.49 0.34 0.18 0.14 23.57 2.24 16,540 2,574 Open in a separate window Sixteen C57BL/10 mice were injected i.v. with 150 g of SEB and killed 48 h later. Lymph node cells were then cultured with either 150.