Small cell lung cancer cell lines were resistant to FasL and TRAIL-induced apoptosis, which could be explained by an absence of Fas and TRAIL-R1 mRNA expression and a deficiency of surface TRAIL-R2 protein. In addition, caspase-8 expression was absent, whereas FADD, FLIP and caspases-3, -7, -9 and -10 could be detected. Analysis of SCLC tumors revealed reduced levels of Fas, TRAIL-R1 and caspase-8 mRNA compared to non-small cell lung cancer (NSCLC) tumors. Methylation-specific PCR demonstrated methylation of CpG islands of the Fas, TRAIL-R1 and caspase-8 genes in SCLC cell lines and tumor samples, whereas NSCLC samples were not methylated. Cotreatment of SCLC cells with the demethylating agent 5 0 -aza-2-deoxycytidine and IFNc partially restored Fas, TRAIL-R1 and caspase-8 expression and increased sensitivity to FasL and TRAIL-induced death. These results suggest that SCLC cells are highly resistant to apoptosis mediated by death receptors and that this resistance can be reduced by a combination of demethylation and treatment with IFNc.

No abstract

The inhibitor of apoptosis protein (IAP) survivin is overexpressed in many tumors but is absent in most normal adult tissues. We report high levels of survivin expression in small cell lung cancer (SCLC), and describe the role of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in survivin upregulation. Moreover, the cytoprotective function of survivin in response to the anti-cancer agent cisplatin (CDDP) was investigated. Negative modulation of PI3K/ Akt using pharmacological inhibitors or dominant negative Akt (DN-Akt) decreased Akt kinase activity and resulted in decreased survivin expression and phosphorylation on Thr34, whereas transfection of constitutively active Akt (CA-Akt) increased survivin expression and phosphorylation. Interestingly, we found that treatment of SCLC cells with CDDP further increased survivin expression in a cell cycle independent manner by activation of Akt. CA-Akt or lentiviral survivin also inhibited apoptosis induced by CDDP, whereas DN-Akt or survivin-specific RNA interference sensitized cells to CDDP. We identified survivin as an anti-apoptotic protein in SCLC cells that is regulated by Akt, and demonstrate that treatment with the DNA damaging agent CDDP activates the PI3K/Akt/survivin pathway that in part protects cells from drug-induced apoptosis. ' 2005 Wiley-Liss, Inc.

Our study was designed to investigate the role of the anti-apoptotic proteins Bcl-2 and Bcl-xL in the chemoresistance of cells derived from malignant pleural mesothelioma. First, we determined the basal expression levels of Bcl-2 and Bcl-xL in mesothelioma cells and examined the effect of their downregulation by antisense oligonucleotides. Bcl-xL mRNA and protein could be readily detected in mesothelioma cell lines, whereas only low levels of Bcl-2 mRNA and protein were found. Preferential downregulation of either Bcl-xL alone or of Bcl-xL and Bcl-2 simultaneously was achieved by treatment with antisense oligonucleotides 4259 and 4625, respectively, whereas the expression of other apoptosis-relevant genes remained unaffected. Treatment with oligonucleotides 4259 or 4625 lowered the apoptosis threshold in ZL34 mesothelioma cells, as indicated by an increase in cell death accompanied by increased caspase-3-like activity, a decrease of the mitochondrial transmembrane potential and the cleavage of procaspase-7 and ICAD. In addition to the direct induction of apoptosis, antisense treatment sensitized ZL34 cells to the cytostatic effect of cisplatin and gemcitabine, with the combination of 4625 and cisplatin being the most effective. Our results demonstrate that Bcl-2 and Bcl-xL antisense treatment facilitates apoptosis in mesothelioma cells and suggest the use of Bcl-2/Bcl-xL bispecific antisense treatment in combination with cisplatin or gemcitabine for therapy of malignant pleural mesothelioma.

Malignant pleural mesotheliomas (MPMs) are usually wild type for the p53 gene but contain homozygous deletions in the INK4A locus that encodes p14(ARF), an inhibitor of p53-MDM2 interaction. Previous findings suggest that lack of p14(ARF) expression and the presence of SV40 large T antigen (L-Tag) result in p53 inactivation in MPM. We did not detect SV40 L-Tag mRNA in either MPM cell lines or primary cultures, and treatment of p14(ARF)-deficient cells with cisplatin (CDDP) increased both total and phosphorylated p53 and enhanced p53 DNA-binding activity. On incubation with CDDP, levels of positively regulated p53 transcriptional targets p21(WAF), PIG3, MDM2, Bax, and PUMA increased in p14(ARF)-deficient cells, whereas negatively regulated survivin decreased. Significantly, p53-induced apoptosis was activated by CDDP in p14(ARF)-deficient cells, and treatment with p53-specific siRNA rendered them more CDDP-resistant. p53 was also activated by: 1) inhibition of MDM2 (using nutlin-3); 2) transient overexpression of p14(ARF); and 3) targeting of survivin using antisense oligonucleotides. However, it is noteworthy that only survivin downregulation sensitized cells to CDDP-induced apoptosis. These results suggest that p53 is functional in the absence of p14(ARF) in MPM and that targeting of the downstream apoptosis inhibitor survivin can sensitize to CDDP-induced apoptosis.