Proteinase-3, a serine protease which mediates doxorubicin-induced apoptosis in the HL-60 leukemia cell line, is downregulated in its doxorubicin-resistant variant.
We report here that expression of proteinase 3 (PR3), a serine protease, is down-regulated in the HL60/ADR multidrug resistant variant of the human myelogenous leukemia cell line HL-60, and that down-regulation of PR3 is associated with doxorubicin (DOX) resistance in these cells. To determine whether PR3 is involved in DOX-induced apoptosis in HL-60 cells, and whether its loss causes resistance to DOX, we inhibited PR3 expression by an anti-sense PR3 oligodeoxynucleotide and showed that inhibition of PR3 expression results in a significant reduction in DOX-induced DNA fragmentation and increased resistance to DOX-induced apoptosis. Our results revealed that PR3-mediated DOX-induced apoptosis in HL-60 cells is independent of the loss of mitochondrial membrane potential (deltapsi(m)) and activation of the caspase-8 and -9 pathways. Moreover, while PR3 is involved in the cleavage of caspase-3, PR3-mediated DOX-induced DNA fragmentation and apoptosis were not prevented by a specific inhibitor of caspase-3. These data suggest that activation of caspase-3 alone is not sufficient to trigger PR3-mediated DOX-induced apoptosis. Treatment with an anti-PR3 oligomer significantly decreased reactive oxygen species (ROS) generation in cells treated with low concentrations of DOX, revealing a role for PR3 in enhancing production of DOX-induced ROS. Moreover, DOX-induced apoptosis at 0.001-0.01 microM was only inhibited in HL-60 cells pre-treated with the antioxidant N-acetyl-cysteine in the absence of anti-PR3, revealing that DOX-induced apoptosis in these cells is PR3- and ROS-dependent. Our results show that PR3 is involved in DOX-induced ROS-dependent apoptosis and that its loss is associated with resistance to DOX in HL-60 cells.
Department of Pharmacology and Toxicology, Indiana University, 1044 West Walnut R4-119, Indianapolis, Indiana, IN 46202, USA.
Ching-Huang
CH
Wu
John
J
Gordon
Mojgan
M
Rastegar
Besim
B
Ogretmen
Ahmad R
AR
Safa
D017209:Apoptosis / Q000187:drug effects*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D053938:DNA Fragmentation; D015536:Down-Regulation; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D018922:HL-60 Cells; D006801:Humans; D020287:In Situ Nick-End Labeling; D020128:Inhibitory Concentration 50; D007425:Intracellular Membranes / Q000378:metabolism; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D053146:Myeloblastin; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D061945:Caspase Inhibitors; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D004317:Doxorubicin; D012697:Serine Endopeptidases; D053146:Myeloblastin; D020169:Caspases