Constitutively activated Stat3 protects fibroblasts from serum withdrawal and UV-induced apoptosis and antagonizes the proapoptotic effects of activated Stat1 - PubMed (original) (raw)
Constitutively activated Stat3 protects fibroblasts from serum withdrawal and UV-induced apoptosis and antagonizes the proapoptotic effects of activated Stat1
Y Shen et al. Proc Natl Acad Sci U S A. 2001.
Abstract
Stats1 and 3 (signal transducers and activators of transcription) can be activated simultaneously, although not necessarily to the same degree or duration, by the interaction of cells with the same polypeptide ligand (EGF, PDGF, or high concentrations of IL-6, for example). However, these two Stat proteins can mediate opposing effects on cell growth and survival. Stat1 activation slows growth and promotes apoptosis. In contrast, activated Stat3 can protect cells from apoptosis. Furthermore, a constitutively active form of Stat3, Stat3-C (bridged by S-S linkages between cysteines instead of phosphotyrosines) can induce cellular transformation of fibroblasts. We have determined that fibroblasts transformed by Stat3-C are more resistant to proapoptotic stimuli than nontransformed cells. Also, to examine the potential opposing roles in apoptosis of Stat1 and Stat3, we studied the cervical carcinoma-derived cell line, Me180, which undergoes Stat1-dependent, IFN gamma-induced apoptosis. Me180 cells that express Stat3-C are protected against IFN gamma-mediated apoptosis.
Figures
Figure 1
Constitutively active Stat3-C and v-src inhibit apoptosis in fibroblast cells. (A) 3T3 and derived cell lines were serum starved, and apoptosis was assayed at the indicated time points by FACs quantitation of propidium iodide-stained cells in the subG1 population. (B) 3T3 and derived cell lines were serum starved, and apoptosis was assayed at the indicated time points by assaying extranuclear DNA fragmentation, as described in Materials and Methods. This method extracts both extranuclear RNA and DNA. After RNase treatment, the extranuclear DNA was electrophoresed in a 1.5% agarose gel and visualized by UV fluorescence after staining by ethidium bromide. As a loading control, the purified nucleic acids without RNase treatment were similarly analyzed by agarose gel electrophoresis and ethidium bromide staining. Jurkat cells induced into apoptosis by camptothecin are shown as controls for DNA laddering. (C) 3Y1 and derived cells were treated with UV radiation, and apoptosis was assayed 24 h after treatment by FACs quantitation of propidium iodide-stained cells in the subG1 population. A representative experiment was shown with the mean + standard deviation of the triplicate samples.
Figure 2
Me180 cells are growth inhibited by low concentrations of IFNγ. (A) 1 × 104 Me180 cells were plated onto 3-cm dishes. Once the cells had adhered (≈12 h later), IFNγ (0.5 ng/ml), IL-6 (200 units/ml), both, or neither were added to the adherent cells grown in complete media. On a daily basis, the cells were counted by trypan blue exclusion (the mean of triplicate samples). (B) 1 × 106 cells were plated onto 6-cm dishes and treated with or without IFNγ (0.5 ng/ml) for 24 h. The cells were harvested, stained with propidium iodide (PI), and analyzed by FACS. The number of cells in G1, S, and G2 was determined. (C) Me180 cells were grown and treated with either IFNγ (0.5 ng/ml), IL-6 (200 units/ml), or both for 1, 4, 12, or 24 h. Cells were harvested and nuclear extracts were isolated from the cell samples. Equal amounts of protein were incubated with radiolabeled double stranded m67(binding site for Stat1 and or Stat3) and resolved on a nondenaturing acrylamide gel.
Figure 3
Me180 cells expressing Stat3-C. (A) Stable clones of Me180 cells were isolated expressing either a control plasmid or a Stat3-C expressing plasmid. The clones were maintained in G418 containing media. Nuclear extracts were isolated from these clones and one example is shown here. Ten micrograms of protein from each cell line (3T3, 3T3–3C, Me, Me3-C) was resolved on an SDS polyacrylamide gel, transferred to nitrocellulose, and probed with anti-FLAG antisera. Two micrograms of nuclear extract from the same cell lines (see above) were incubated with radiolabeled m67 and resolved on a nondenaturing EMSA gel. (B) Parental Me180 cells (Me) and Me180 expressing Stat3-C cells (Me 3-C) were either left untreated (serum containing media) or treated with IFNγ (0.5 ng/ml) for 0.5, 1, 2, or 6 h. Nuclear extracts were isolated from these, incubated with radiolabeled m67, and resolved on an EMSA gel.
Figure 4
Me180 cells expressing Stat3-C are protected from IFNγ-induced apoptosis. (A) 2 × 104 Me180 cells stably transfected with the control plasmid RcCMV [Me(Rc)] and Me180 expressing Stat3-C cells (Me3-C) were plated onto 3-cm dishes. Once the cells had adhered (≈12 h later), IFNγ (0.5 ng/ml, 1.5 ng/ml, or 5 ng/ml) was added to the adherent cell culture. Then, 72 h later, the cells were washed with PBS and photographed by using phase microscopy. (B) As described above. Cells were counted on a daily basis by trypan blue exclusion (the mean of triplicate samples). (C) Me(Rc) and Me(3-C) cells were treated at the indicated IFNγ concentration (ng/ml) for 48 h, and apoptosis was assayed by FACs quantitation of propidium iodide-stained cells in the subG1 population. Whenever indicated, TNF-α was added to a concentration of 5 μg/ml. Shown are representative results of four independent experiments. (D) Me(Rc) and Me3-C cells were treated with IFNγ (18 ng/ml) for 12 and 24 h, extranuclear DNA was prepared and analyzed by agarose gel electrophoresis and ethidium bromide staining, as described in Materials and Methods.
Figure 5
Me180 cells expressing Stat3-C contain elevated expression of Bcl-xL and survivin, and decreased expression of Bax. (A) Northern blot analysis of Bcl-xL on total RNA prepared from Me180 cells (Me) or Me180 cells stably expressing Stat3C (Me 3-C) treated with IFN-γ for 4 h (IFN-γ) or untreated (−). Blot was reprobed with GAPDH to control equal loading. (B) Western blot analysis of Bcl-2, Bax, and survivin. Fifty micrograms of whole cell extract from Me180 cells stably transfected with the control plasmid [Me(Rc)] or Me180 cells expressing Stat3C (Me 3-C) treated with IFN-γ (18 ng/ml) for 24 h (IFN-γ) or untreated (−) were blotted with the indicated antibody.
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