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The CrmA- and TPCK-sensitive pathways that trigger oligonucleosome-sized DNA fragmentation in camptothecin-induced
A T Sané1, E Schmitt, A Steyaert
1Louis-Charles Simard Research Center, Montreal Cancer Institute, CHUM, QC, Canada.
Abstract:
Abstract: In human B lymphoma Namalwa variant cells expressing the serpin-like CrmA protein, the kinetics of oligonucleosome-sized DNA fragmentation was retarded compared with that of control Namalwa cells following camptothecin treatment. However, no difference in the kinetics of high molecular weight DNA fragmentation was observed between the two lines after camptothecin treatment. Similar delay and inhibition of the oligonucleosome-sized DNA fragmentation was observed in human B lymphoma Namalwa and monocytic-like leukemia U-937 cells coincubated in the presence of various concentrations of N-tosyl-L-phenylalanyl chloromethylketone and camptothecin. The effect of N-tosyl-L-phenylalanyl chloromethylketone was similar to that of CrmA and did not prevent the appearance of high molecular weight DNA fragments. Similar suppression of camptothecin-induced internucleosomal DNA fragmentation was also observed in a cell-free system when cytosolic extracts obtained from camptothecin-treated Namalwa and U-937 cells were coincubated with untreated nuclei in the presence of N-tosyl-L-phenylalanyl chloromethylketone. Furthermore, N-tosyl-L-phenylalanyl chloromethylketone had no significant effects on caspase-3-like activities in camptothecin-treated Namalwa and U-937 cells. Hydrolysis of Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin, a fluorogenic substrate with caspase-3-like activities, was detected in extracts prepared from camptothecin-treated Namalwa and U-937 cells with no apparent difference in the time courses of caspase-3-like activation in the absence or presence of N-tosyl-L-phenylalanyl chloromethylketone. Similarly, N-tosyl-L-phenylalanyl chloromethylketone was a weak inhibitor of caspase-3-like activities in vitro. Taken together, these observations suggest that the pathway sensitive to N-tosyl-L-phenylalanyl chloromethylketone is involved in camptothecin-induced oligonucleosome-sized DNA fragmentation. Furthermore, inhibition of this pathway had no effect on caspase-3-like activation and on the occurrence of high molecular weight DNA fragmentation.
Insights
Camptothecin treatment causes DNA fragmentation in lymphoma cells. A specific pathway, sensitive to N-tosyl-L-phenylalanyl chloromethylketone, influences DNA fragmentation but not caspase-3 activation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Camptothecin is a chemotherapeutic agent that induces DNA fragmentation.
- DNA fragmentation is a hallmark of apoptosis.
- The specific pathways regulating DNA fragmentation are not fully understood.
Purpose of the Study:
- To investigate the role of a specific pathway in camptothecin-induced DNA fragmentation.
- To determine if this pathway affects caspase-3 activation.
Main Methods:
- Studied DNA fragmentation in human B lymphoma Namalwa and monocytic-like leukemia U-937 cells treated with camptothecin.
- Utilized N-tosyl-L-phenylalanyl chloromethylketone, a known inhibitor of a specific pathway.
- Assessed caspase-3-like activities using a fluorogenic substrate.
Main Results:
- N-tosyl-L-phenylalanyl chloromethylketone and CrmA protein inhibited oligonucleosome-sized DNA fragmentation induced by camptothecin.
- High molecular weight DNA fragmentation was not affected by N-tosyl-L-phenylalanyl chloromethylketone.
- N-tosyl-L-phenylalanyl chloromethylketone did not significantly affect caspase-3-like activities in vitro or in cell extracts.
Conclusions:
- A pathway sensitive to N-tosyl-L-phenylalanyl chloromethylketone is involved in camptothecin-induced oligonucleosome-sized DNA fragmentation.
- Inhibition of this pathway does not impact caspase-3 activation or high molecular weight DNA fragmentation.