Reactive Oxygen Intermediates Are Involved in the Induction of CD95 Ligand mRNA Expression by Cytostatic Drugs in Hepatoma Cells

Abstract

Oxidative stress has been associated with the induction of programmed cell death. The CD95 ligand/receptor system is a specific mediator of apoptosis. We have used the model of drug-induced apoptosis to assess whether the CD95 ligand mRNA is induced by reactive oxygen intermediates. Treatment of HepG2 hepatoma cells with bleomycin induced the production of reactive oxygen intermediates and, as an additional parameter of oxidative stress, resulted in glutathione (GSH) depletion. In parallel, CD95 ligand mRNA expression was induced. In a similar fashion CD95 ligand mRNA expression increased after treatment with H 2 O 2 . Additional treatment with the antioxidant and GSH precursor Nacetylcysteine resulted in partial restoration of intracellular GSH levels and in reduced induction of CD95 ligand mRNA. Induction of CD95 ligand mRNA by bleomycin was further reduced by combined treatment with N-acetylcysteine and deferoxamine. These data suggest a direct role of oxygen radicals in the induction of the CD95 ligand.CD95 (APO-1/Fas) is a 45-kDa glycosylated transmembrane protein belonging to the tumor necrosis factor receptor family of type I membrane proteins (1, 2). The CD95 ligand (CD95L) is a 40-kDa Type II transmembrane protein and a member of the tumor necrosis factor family of cytokines (1, 3). In addition to the transmembrane form, a soluble form of the CD95L exists (4). Binding of the CD95L to its receptor CD95 induces apoptosis. The CD95/CD95L system plays a role in the deletion of T lymphocytes in the peripheral immune system, in the shutting off of an immune response, in T lymphocyte-mediated cytotoxicity, and in the elimination of CD95-expressing leukocytes in immune privileged sites (5-10).CD95 signaling occurs through the death-inducing signaling complex and the activation of a cascade of interleukin converting enzyme/Ced3 proteases (11, 12), which are now designated caspases (13). A cell expressing both CD95 and CD95L undergoes suicide or can cause fratricide (5, 6). CD95L is expressed in activated T lymphocytes (3) but its expression can also be induced by cytostatic agents in a variety of different cell lines (14,15). Furthermore, CD95L expression has been observed in hepatocytes in vivo in patients with alcoholic hepatitis (16). Thus, CD95L expression seems to be induced by different mechanisms of cellular injury and might be an important tool for the organism to eliminate damaged cells. Little is known about the exact mechanism of induction of CD95L.The CD95L promoter has been described to contain NF-B binding sites (17). Therefore, induction of CD95L mRNA might involve reactive oxygen species (ROS).1 In line with this assumption is the observation of apoptotic cell death in different cell lines after oxidative stress (18,19). In the present study we have used the model of bleomycin-induced apoptosis to investigate the possible role of ROS in the induction of CD95L mRNA. Bleomycin has been described as a potent inducer of apoptosis, involving up-regulation of CD95 receptor and ligand expression (15). W...

The ATM protein is required for sustained activation of NF-κB following DNA damage

“…This is daring to speculate because both normal and AT transformed lymphoblasts show high constitutive NF-kB activity (data not shown), probably consecutive to maturation in B cells before EBV transformation, but AT lymphoblasts remain hypersensitive to ionizing radiation (Lavin and Shiloh, 1997). In addition, some reports indicate that NF-kB does not a ect or even promote programmed cell death in some systems (Kasibhatla et al, 1998;Hug et al, 1997). Jung et al (1998) recently showed that the N-terminally truncated IkBa protein that complements the AT phenotype in AT5BIVA cells (Jung et al, 1995) diminishes apoptosis in AT cells but enhances apoptosis in normal (MRC5-CV1) ®broblasts.…”

Induction of CD95 ligand and apoptosis by doxorubicin is modulated by the redox state in chemosensitive- and drug-resistant tumor cells

“…These results are in agreement with data recently published showing that bleomycininduced CD95-L mRNA involves ROS in hepatoma cells. 48 Furthermore, downregulation of CD95 expression was not found in chemosensitive cells after pretreatment with NAC or GSH at time points used in these experiments (data not shown). Defective activation of caspases by cytotoxic drugs after pretreatment with NAC or GSH was also reflected by the failure to cleave the prototype caspase substrate poly(ADP-ribose)polymerase (PARP).…”

“…25 Furthermore, it was found, that CD95-L mRNA induction involves ROS in hepatoma cells treated with bleomycin. 48 GSH has also been found to modulate T-cell apoptosis e.g. during HIV infection.…”

DNA-damaging reagents induce apoptosis through reactive oxygen species-dependent Fas aggregation

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