Abstract
We investigated the molecular mechanism of the synergism between interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) documented in a variety of biological occasions such as tumor cell death and inflammatory responses. IFNgamma/TNFalpha synergistically induced apoptosis of ME-180 cervical cancer cells. IFNgamma induced STAT1 phosphorylation and interferon regulatory factor 1 (IRF-1) expression. Transfection of phosphorylation-defective STAT1 inhibited IFNgamma/TNFalpha-induced apoptosis, whereas IRF-1 transfection induced susceptibility to TNFalpha. Dominant-negative IkappaBalpha transfection sensitized ME-180 cells to TNFalpha. IFNgamma pretreatment attenuated TNFalpha- or p65-induced NF-kappaB reporter activity, whereas it did not inhibit p65 translocation or DNA binding of NF-kappaB. IRF-1 transfection alone inhibited TNFalpha-induced NF-kappaB activity, which was reversed by coactivator p300 overexpression. Caspases were activated by IFNgamma/TNFalpha combination; however, caspase inhibition did not abrogate IFNgamma/TNFalpha-induced cell death. Instead, caspase inhibitors directed IFNgamma/TNFalpha-treated ME-180 cells to undergo necrosis, as demonstrated by Hoechst 33258/propidium iodide staining and electron microscopy. Taken together, our results indicate that IFNgamma and TNFalpha synergistically act to destroy ME-180 tumor cells by either apoptosis or necrosis, depending on caspase activation, and STAT1/IRF-1 pathways initiated by IFNgamma play a critical role in IFNgamma/TNFalpha synergism by inhibiting cytoprotective NF-kappaB. IFNgamma/TNFalpha synergism appears to activate cell death machinery independently of caspase activation, and caspase activation seems to merely determine the mode of cell death.
Highlights
We investigated the molecular mechanism of the synergism between interferon ␥ (IFN␥) and tumor necrosis factor ␣ (TNF␣) documented in a variety of biological occasions such as tumor cell death and inflammatory responses
Our results indicate that IFN␥ and TNF␣ synergistically act to destroy ME-180 tumor cells by either apoptosis or necrosis, depending on caspase activation, and STAT1/interferon regulatory factor 1 (IRF-1) pathways initiated by IFN␥ play a critical role in IFN␥/TNF␣ synergism by inhibiting cytoprotective NF-B
We present evidence that STAT1/IRF-1 pathways initiated by IFN␥ play a central role in IFN␥/TNF␣ synergism in the induction of ME-180 cell apoptosis
Summary
Cell Line and Reagents—ME-180 cervical cancer cell line was obtained from ATCC (Manassas, VA) and grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 2 mM glutamine, and penicillin-streptomycin (Life Technologies, Inc.). Caspase-3 or -8 fluorogenic substrates (Ac-DEVD-AMC or Ac-IETD-AMC) were incubated with cytokine-treated cell lysates for 1 h at 37 °C, AMC liberated from Ac-DEVD-AMC or Ac-IETD-AMC was measured using a fluorometric plate reader with an excitation wavelength of 380 nm and an emission wavelength of 420- 460 nm. 48 h after the transfection, cells were treated with cytokines After another 48 h, the cells were fixed with 0.5% glutaraldehyde for 10 min at room temperature and stained with X-gal (5-bromo-4-chloro-3-indolyl -D-galactopyranoside; 1 mg/ml) in 4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, 2 mM magnesium chloride at 37 °C for detection of blue cells. After 5 h, activities of firefly luciferase and Renilla luciferase in transfected cells were measured sequentially from a single sample using the dual-luciferase reporter assay system (Promega). DNA-protein complexes were resolved by electrophoresis in a 5% nondenaturing polyacrylamide gel, dried, and visualized by autoradiography
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