These findings demonstrate that autophagy may amplify apoptosis when associated with a death signaling pathway. They are also evidence that inhibition of autophagy is a novel mechanism of the antiapoptotic function of NF-B activation. We suggest that stimulation of autophagy may be a potential way bypassing the resistance of cancer cells to anti-cancer agents that activate NF-B.

We recently reported that inhibition of NF-kB activation as a consequence of the overexpression of a degradationresistant form of IkBa [IkBa(A32/36)] sensitized Ewing sarcoma cells to TNFa-induced killing. The c-Jun Nterminal kinases (JNK) have been shown to participate in death signaling triggered by certain stimuli and are activated by TNFa. To obtain insight into the mechanism of the anti-apoptotic e ect of NF-kB, we compared the pro®les of JNK activation by TNFa in control cells and in cells in which NF-kB activation was impaired. We show here that JNK activation was transient in control cells but remained elevated in IkBa(A32/36)-expressing cells. NF-kB repressed speci®cally the JNK pathway, since the kinetics of activation of the other TNFaactivated-MAP kinase p38 were identical in both cells. Prolongation of JNK activation in IkBa(A32/36)-expressing cells was not inhibited by the broad spectrum caspase inhibitor Z-VAD-FMK and thus was not the consequence of caspase activation. Pretreatment of control cells with the phosphatase inhibitor vanadate greatly prolonged JNK activation by TNFa and resulted in induction of apoptosis by this cytokine. Moreover, overexpression of a dominant-negative mutant of JNK1 decreased TNFa-induced apoptosis in cells expressing the super repressor of NF-kB, indicating that the sustained activation of JNK1 participated in death signaling triggered by TNFa. Our results provide evidence that the repression of JNK activation by NF-kB participates in the anti-apoptotic e ect of this transcription factor in TNFa-treated Ewing sarcoma cells. Oncogene (2001) 20, 4365 ± 4372.

p21WAF1 appears to be a major determinant of the cell fate in response to anticancer therapy. It was shown previously that HCT116 human colon cancer cells growing in vitro enter a stable arrest upon DNA damage, whereas cells with a defective p21 WAF1 response undergo apoptosis. Here we report that the enhanced sensitivity of HCT116/p21 ؊/؊ cells to chemotherapeutic drug-induced apoptosis correlates with an increased expression of p53 and a modification of their Bax/Bcl-2 ratio in favor of the pro-apoptotic protein Bax. Treatment of HCT116/p21؊/؊ cells with daunomycin resulted in a reduction of the mitochondrial membrane potential and in activation of caspase-9, whereas no such changes were observed in HCT116/p21 ؉/؉ cells, providing evidence that p21 WAF1 exerts an antagonistic effect on the mitochondrial pathway of apoptosis. Moreover, the role of p53 in activation of this pathway was demonstrated by the fact that inhibition of p53 activity by pifithrin-␣ reduced the sensitivity of HCT116/p21 ؊/؊ cells to daunomycin-induced apoptosis and restored a Bax/Bcl-2 ratio similar to that observed in HCT116p21 ؉/؉ cells. Enhancement of p53 expression after disruption of p21 WAF1resulted from a stabilization of p53, which correlated with an increased expression of the tumor suppressor p14 ARF , an inhibitor of the ubiquitin ligase activity of Mdm2. In accordance with the role of p14 ARF in p53 stabilization, overexpression of p14 ARF in HCT116/ p21 ؉/؉ cells resulted in a strong increase in p53 activity. Our results identify a novel mechanism for the antiapoptotic effect of p21 WAF1 consisting in maintenance of mitochondrial homeostasis that occurs in consequence of a negative control of p14 ARF expression.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective transepithelial Cl- transport. The regulation of CF gene expression is not fully understood. We report that interferon-gamma (IFN-gamma), but not IFN-alpha or -beta, downregulates CFTR mRNA levels in two colon-derived epithelial cell lines, HT-29 and T84, in a time- and concentration (from 0.1 IU/ml)-dependent manner. IFN-gamma has no effect on the transcription rate of the CFTR gene but reduces CFTR mRNA half-life, indicating that it exerts a posttranscriptional regulation of CFTR expression, at least partly, through destabilization of the transcripts. Cells treated with IFN-gamma contain subnormal amounts of 165-kDa CFTR protein. Assays of adenosine 3',5'-cyclic monophosphate-stimulated 36Cl- efflux and whole cell currents show that CFTR function is diminished in IFN-gamma-treated cells. IFN-gamma and tumor necrosis factor-alpha synergistically reduce CFTR gene expression. Our results suggest that production of these cytokines in response to bacterial infections and inflammatory disorders may alter transmembrane Cl- transport.

No abstract

Repression of activation of c-Jun N-terminal kinase (JNK) participates in the anti-apoptotic effect of nuclear factor-jB (NF-jB) in TNFa-treated Ewing sarcoma cells. As oxidative stress is one of the most prominent activators of JNK, we investigated the relationship between TNFa-induced NF-jB activation and the control of oxidative stress. Inhibition of NFjB activation resulted in an increase in TNFa-induced ROS production, lipid peroxidation and protein oxidation. Those ROS and lipid peroxides were both involved in TNFa-induced apoptosis, whereas only ROS elevation triggered sustained JNK activation. TNFa increased the level of two antioxidant enzymes, thioredoxin and manganese superoxide dismutase by an NFjB-dependent mechanism. Inhibition of expression or activity of these enzymes sensitized cells to TNFa-induced apoptosis, indicating their functional role in protection from cell death. Thus, agents that inhibit activities of these enzymes may prove helpful in the treatment of Ewing tumors.

No abstract

The Ewing sarcoma is the second most common bone tumor in children and young adults. Despite the advances in therapy, the 5-year survival rate for patients with metastatic disease is poor, indicating the need for alternative treatments. Here, we report that 2-methoxyestradiol (2-Me), a natural estrogen metabolite, induced a caspase-dependent apoptosis of Ewing sarcoma-derived cells independently of their p53 status. 2-Me-induced apoptosis occurred through the mitochondrial death pathway as evidenced by reduction of the mitochondrial transmembrane potential, cytochrome c release and caspase-9 activation. Treatment of cells with 2-Me resulted in generation of intracellular H 2 O 2 , which occurred earlier than caspase-9 activation. The H 2 O 2 -reducing agent Ebselen and the lipid peroxidation inhibitor vitamin E decreased both 2-Me-induced caspase-9 activation and cell death, thus providing evidence for a role of H 2 O 2 and lipid peroxides in the initiation of this process. Rotenone, an inhibitor of the mitochondrial respiratory chain, abolished both apoptosis and H 2 O 2 production, thereby identifying mitochondria as the source of H 2 O 2 . Moreover, we observed that treatment of cells with 2-Me or H 2 O 2 induced activation of the c-Jun N-terminal kinase (JNK). Overexpression of a dominantnegative mutant of JNK1 reduced 2-Me-induced apoptosis indicating that JNK participates in this process. Altogether, our results provide evidence that 2-Me triggers apoptosis of Ewing sarcoma cells through induction of a mitochondria redox-dependent mechanism and suggest that this compound or other agents that selectively increase the level of reactive oxygen species may prove useful to the development of novel strategies for treatment of Ewing tumors.