Proteinase-3, a serine protease which mediates doxorubicin-induced apoptosis in the HL-60 leukemia cell line, is downregulated in its doxorubicin-resistant variant

Ching Huang Wu, John Gordon, Mojgan Rastegar, Besim Ogretmen, Ahmad Safa

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

We report here that expression of proteinase 3 (PR3), a serine protease, is down-regulated in the HL60/ADR mulfidrug 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 (ΔΨm) 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 μM 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.

Original languageEnglish
Pages (from-to)5160-5174
Number of pages15
JournalOncogene
Volume21
Issue number33
DOIs
StatePublished - Aug 1 2002

Fingerprint

Myeloblastin
HL-60 Cells
Serine Proteases
Doxorubicin
Leukemia
Down-Regulation
Apoptosis
Cell Line
Reactive Oxygen Species
Caspase 3
DNA Fragmentation
Myeloid Leukemia
Caspase 9
Caspase 8
Mitochondrial Membrane Potential
Oligodeoxyribonucleotides

Keywords

  • Apoptosis
  • Caspases
  • Doxorubicin
  • Leukemia
  • Multidrug resistance
  • Proteinase-3

ASJC Scopus subject areas

  • Molecular Biology
  • Cancer Research
  • Genetics

Cite this

Proteinase-3, a serine protease which mediates doxorubicin-induced apoptosis in the HL-60 leukemia cell line, is downregulated in its doxorubicin-resistant variant. / Wu, Ching Huang; Gordon, John; Rastegar, Mojgan; Ogretmen, Besim; Safa, Ahmad.

In: Oncogene, Vol. 21, No. 33, 01.08.2002, p. 5160-5174.

Research output: Contribution to journalArticle

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abstract = "We report here that expression of proteinase 3 (PR3), a serine protease, is down-regulated in the HL60/ADR mulfidrug 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 (ΔΨm) 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 μM 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.",
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