David M. Tillman

Reactive Oxygen Species Regulate Caspase Activation in Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Resistant Human Colon Carcinoma Cell Lines

The effects of reactive oxygen species (ROS) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in solid cancers have yet to be clearly defined. In this study, we found that the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a reduction in DeltaPsim and generation of ROS. This uncoupling effect enhanced TRAIL-induced apoptosis in TRAIL-resistant human colon carcinoma cell lines (RKO, HT29, and HCT8). Sensitization was inhibited by benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone, indicating the requirement for caspase activation. CCCP per se did not induce apoptosis or release of proapoptotic factors from mitochondria. Generation of ROS by CCCP was responsible for TRAIL-induced Bax and caspase activation because scavenging ROS completely abrogated apical caspase-8 activation and further downstream events leading to cell death. Overexpression of Bcl-2 did not prevent the initial loss of DeltaPsim and ROS generation following CCCP treatment, but did prevent cell death following TRAIL and CCCP exposure. Uncoupling of mitochondria also facilitated TRAIL-induced release of proapoptotic factors. X-linked inhibitor of apoptosis overexpression abrogated TRAIL-induced apoptosis in the presence of CCCP and decreased initiator procaspase-8 processing, indicating that additional processing of caspase-8 required initiation of a mitochondrial amplification loop via effector caspases. Of interest, depletion of caspase-9 in RKO cells did not protect cells from TRAIL/CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9-independent pathway. Data suggest that in the presence of mitochondrial-derived ROS, TRAIL induced mitochondrial release of Smac/DIABLO and inactivation of X-linked inhibitor of apoptosis through caspase-9-independent activation of caspase 3.

Hypermethylation of the gene promoter and enhancer region can regulate Fas expression and sensitivity in colon carcinoma

AbstractExpression of the cell surface receptor Fas is frequently lost or decreased during tumor progression in human colon carcinomas. The methylation status of a 583 bp CpG-rich region within the Fas promoter (−575 to +8) containing 28 CpG sites was determined in human colon carcinoma cell lines. In Caco2 (no Fas expression), 82–93% of CpG sites were methylated, whereas none were methylated in GC3/c1 (high Fas expression). In RKO (intermediate level of Fas), a single CpG site, located at –548, was 100% methylated. The inhibitor of methylation, 5-aza-2′-deoxycytidine (5-azadC), upregulated Fas expression in four of eight cell lines, and sensitized RKO cells to recombinant FasL-induced apoptosis. The p53-binding region in the first intron of the Fas gene was partially methylated in Caco2, and 5-azadC potentiated Ad-wtp53-induced upregulation of Fas expression. Methylation-specific PCR of the first intron detected partial methylation in four out of 10 colon carcinoma tumor samples in vivo. The data suggest that DNA hypermethylation is one mechanism that contributes to the downregulation of Fas expression and subsequent loss of sensitivity to Fas-induced apoptosis in colon carcinoma cells.

Expression of genes that regulate Fas signalling and Fas-mediated apoptosis in colon carcinoma cells.

The expression of genes that regulate Fas-induced apoptosis has been examined in 10 human cultured colon carcinoma cell lines with defined and varied sensitivity to the cytolytic anti-Fas MoAb CH-11. Four lines demonstrated sensitivity to CH-11 (HT29, GC3/c1, TS−, Thy4), and six were resistant to the induction of apoptosis vis Fas. In nine lines expressing Fas, PCR-sequencing indicated that the death domain contained wt sequences. Downstream of Fas, expression of FADD/MORT1 and FLICE, essential components of the DISC, and negative regulators of Fas signalling including sFas, FAP-1 and Bcl-2, showed no correlation between levels of expression and sensitivity to Fas-mediated cytotoxicity. However, levels of the Fas antigen varied by >1000-fold, and correlated with CH-11 sensitivity. Following fourfold elevation in Fas expression in HT29 cells treated with interferon-γ, a synergistic effect on Fas-mediated apoptosis was obtained when CH-11 and interferon-γ were combined.

A caspase-8-independent component in TRAIL/Apo-2L-induced cell death in human rhabdomyosarcoma cells.

AbstractTumor necrosis factor related apoptosis inducing ligand (TRAIL) belongs to the Tumor necrosis factor (TNF) family of death-inducing ligands, and signaling downstream of TRAIL ligation to its receptor(s) remains to be fully elucidated. Components of the death-inducing signaling complex (DISC) and TRAIL signaling downstream of receptor activation were examined in TRAIL - sensitive and -resistant models of human rhabdomyosarcoma (RMS). TRAIL ligation induced DISC formation in TRAIL-sensitive (RD, Rh18, Rh30) and TRAIL-resistant RMS (Rh28, Rh36, Rh41), with recruitment of FADD and procaspase-8. In RD cells, overexpression of dominant-negative FADD (DNFADD) completely abolished TRAIL-induced cell death in contrast to dominant-negative caspase- 8 (DNC8), which only partially inhibited TRAIL-induced apoptosis, growth inhibition, or loss in clonogenic survival. DNC8 did not inhibit the cleavage of Bid or the activation of Bax. Overexpression of Bcl-2 or Bcl-xL inhibited TRAIL-induced apoptosis, growth inhibition, and loss in clonogenic survival. Bcl-2 and Bcl-xL, but not DNC8, inhibited TRAIL-induced Bax activation. Bcl-xL did not inhibit the early activation of caspase-8 (<4 h) but inhibited cleavage of Bid, suggesting that Bid is cleaved downstream of the mitochondria, independent of caspase-8. Exogenous addition of sphingosine also induced activation of Bax via a caspase-8-and Bid-independent mechanism. Further, inhibition of sphingosine kinase completely protected cells from TRAIL-induced apoptosis. Data demonstrate that in RMS cells, the TRAIL signaling pathway circumvents caspase-8 activation of Bid upstream of the mitochondria and that TRAIL acts at the level of the mitochondria via a mechanism that may involve components of the sphingomyelin cycle.