Reply

We appreciate the careful and critical reading of our paper by Kruyt et al. (1) and the opportunity to respond to their comments. Prior to our report, several researchers had indicated the defect of the apoptosome pathway, a common execution mechanism of apoptosis, in cancer cells, which could confer resistance to chemotherapy. Therefore, the initial purpose of our study was to confirm and further identify the factors responsible for this defect in human NSCLC 1 cells. We found that the apaf-1/caspase-9-mediated pathway was significantly down-regulated in H460 cells, a finding consistent with that of Ferreira et al. (2) showing that the caspase-9-mediated pathway does not function in cisplatin-induced apoptosis of H460 cells. We further tested the effect of the Smac-N7 peptide on apoptosome activation and found that it effectively enhanced that activity. We indicated the involvement of IAP in apoptosis inhibition in the cells by testing the effect of a cell-permeable version of the peptide on caspase activation and apoptosis. Our results, however, do not exclude the possible involvement of other inhibitory factors, especially upstream of caspase-9 cleavage. Contrary to the above comment that indicates caspase-9 cleavage did not occur during cisplatin-induced apoptosis of H460 cells, we detected cytochrome c release as well as weak, but apparent, cleavage of caspase-9 (at Asp315 site) after drug treatment (2 μg/ml for 48 h). 2 This discrepancy could be attributable to the difference in concentration of the drug used or the antibodies that detect caspase-9 cleavage. Moreover, we observed the increased cleavage of caspase-3, but not of caspase-9, when cells were treated with cisplatin together with Smac-N7R8. Important to note is that cotreatment with the Smac-N7R8 peptide, which neutralizes the IAP function, induced elevated caspase-9 (LEHD-cleavage) activity without affecting caspase-9 processing in the cells. 2 These results indicate that weak cleavage of caspase-9 actually occurs after drug treatment in the cells, and IAP suppresses apoptosis induction downstream of caspase-9 cleavage.

On the other hand, we agree with Kruyt et al. (1) that caspase-9 cleavage is significantly weak in H460 cells after cisplatin exposure, suggesting the involvement of some critical factors acting upstream of caspase-9 that inhibit the activation of the apoptosome pathway. Our data also showed that, even in the absence of the Smac peptide, cisplatin induced caspase-3 activation, which could occur through a novel bypassing pathway that involves caspase-8, as reported previously (2) .

Furthermore, Kruyt et al. (1) pointed out the difference between the two studies in sensitivity to cisplatin in H460 cells. Although we could not easily compare the results because of differences in the methods as well as in the time points at which the cell death induction was examined, the discrepancy could be attributed to some differences in cell culture conditions, including serum factors. Indeed, previous reports have indicated that several growth factor signals affect cell survival and apoptosis both upstream and downstream of the apoptosome pathway. Finally, as Kruyt et al. (1) described in their letter, we also observed the frequent overexpression of IAP proteins in NSCLC patient samples. 2 These data indicate that IAP proteins are good and selective targets to potentiate the effect of chemotherapy on NSCLC, whereas their predictive value in the clinic is still open to question. Additional studies are needed to substantiate these views.

• ©2003 American Association for Cancer Research.