The effect of inhibiting topoisomerase I and II on the anti-apoptotic response associated with pro-inflammatory crystals of calcium pyrophosphate dihydrate in human neutrophils.
OBJECTIVE
To investigate the ability of various topoisomerase I and II inhibitors to reverse the pro-survival effects of calcium pyrophosphate dihydrate (CPPD) crystals on human neutrophils, thereby identifying potential agents that may promote the resolution of neutrophil accumulation typical of crystal associated inflammatory diseases.


METHODS
Freshly isolated human neutrophils incubated in the presence of CPPD crystals, with or without the pro-apoptotic cytokine TNF-alpha, were pre-incubated in the presence or absence of the topoisomerase I inhibitors camptothecin, nogalamycin or beta-lapachone, or topoisomerase II inhibitors etoposide, doxorubicin or mitoxantrone. Neutrophil respiratory burst was assessed via chemiluminescence, and two quantitative methods were used for the determination of neutrophil apoptosis; cytoplasmic histone-associated-DNA fragmentation assessment, and endogenous caspase 3 substrate (Ac-DEVD-AMC) cleavage.


RESULTS
Beta-lapachone and mitoxantrone effectively repressed CPPD crystal associated respiratory burst, whereas the other topoisomerase inhibitors had no inhibitory or stimulatory effect. Camptothecin and all of the topoisomerase II inhibitors induced neutrophil apoptosis, even in the presence of the CPPD crystals that normally repress TNF-alpha-induced and spontaneous apoptosis.


CONCLUSIONS
These results suggest that although topoisomerase II antagonists are distinctively effective agents at reversing the pro-survival effects of crystals on neutrophils, camptothecin was unique as a topoisomerase I inhibitor in that it was significantly more effective as a pro-apoptosis inducer than the topoisomerase II poisons without affecting normal neutrophil activation responses.
Tudan
C
C
Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, B.C., Canada V6T 1Z3.
Jackson
J K
JK
Higo
T T
TT
Burt
H M
HM
D017209:Apoptosis / Q000187:drug effects*; D002131:Calcium Pyrophosphate / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D003460:Crystallization; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D009504:Neutrophils / Q000187:drug effects* / Q000201:enzymology; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000494:pharmacology
D004791:Enzyme Inhibitors; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D014409:Tumor Necrosis Factor-alpha; D002131:Calcium Pyrophosphate