According to current understanding, caspase-6 is an effector caspase that is activated downstream of caspase 3 during apoptosis1, 2 and is essential only for the apoptotic cleavage of lamin A in nuclei that express this polypeptide.3 On the other hand, several recent observations have raised the possibility that caspase 6 might also play an important upstream role during anticancer drug-induced apoptosis. First, based on the ability of IETD-fmk but not crmA or caspase 8 deficiency to inhibit resveratrol-induced apoptosis in human T-cell leukemia cell lines, it has been suggested that caspase 6 is the initiator caspase after treatment with this experimental anticancer agent.4 Second, DU145 human prostate cancer cells selected for camptothecin resistance were observed to have diminished expression of procaspase-6.5 More recently, experiments designed to measure procaspase polypeptide levels6 in the 60 human tumor cell lines utilized by the National Cancer Institute to screen potential anticancer drugs7, 8 demonstrated a striking correlation between procaspase 6 polypeptide levels and ability of certain agents to induce cell death (LC50) across the cell lines (Figure 1a and molecular target MT1646 at http://www.dtp.nci.nih.gov/webdata.html). Collectively, these observations raised the possibility that caspase 6 might play a previously unsuspected critical role in the induction of apoptosis after treatment with selected anticancer drugs.
To test this hypothesis, we examined the ability of a number of agents to induce apoptosis in parental and caspase-6−/− DT40 chicken lymphoma cells.3 Our data revealed that the ability of the topoisomerase II poison etoposide to induce apoptosis in the caspase-6−/− cell line was intact (Figure 1b) despite the absence of detectable procaspase-6 protein (Figure 1c), ruling out the possibility that the gene targeting and selection process had raised the apoptotic threshold in this cell line. When treated with camptothecin, the parental and caspase-6−/− cells displayed roughly equal sensitivity to the induction of apoptosis as assessed by the appearance of cells with subdiploid DNA content after propidium iodide staining (Figure 1d) or the activation of caspase 3-like activity (Figure 1e).
Among the compounds that showed a high correlation between procaspase-6 expression and LC50, several were not available for testing (not shown) or failed to induce apoptosis in DT40 cells after 48 h of continuous exposure to the highest tested concentration (Figure 1a). Three of the agents, however, readily induced apoptosis in DT40 cells (Figure 1a and f). Of these three, two (NSC 349856 and NSC 682298) induced slightly more apoptosis in caspase-6−/− DT40 cells than parental cells (Figure 1f). In short, the loss of caspase-6 does not appear to impair the ability of any of these agents to induce apoptosis. Even though the correlations between caspase-6 content and drug sensitivity across 60 cell lines were striking and highly statistically significant for several compounds (Figure 1a), we found no mechanistic basis for these correlations and no evidence that caspase 6 plays an essential role in the cytotoxic response to these agents.
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We thank Dominic Scudiero, Edward Sausville and Susan Holbeck for their assistance in identifying the correlation between procase-6 expression and sensitivity of the 60 cell lines to various agents. Work in our laboratories is supported by Grant CA69008 from the National Cancer Institute.
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Loegering, D., Ruchaud, S., Earnshaw, W. et al. Evaluation of the role of caspase-6 in anticancer drug-induced apoptosis. Cell Death Differ 13, 346–347 (2006). https://doi.org/10.1038/sj.cdd.4401791