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

Oncogene volume 21, pages 5160–5174 (2002)Cite this article

Abstract

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 (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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Acknowledgements

We would like to thank Dr Mary D Kraeszig for her editorial assistance, and also to Drs Edward Srour and Susan Rice for their great advice and help with the flow cytometric analysis. We are very grateful to Dr Ulrich Specks (Mayo Clinic, Rochester, MN, USA) for providing us with the MCPR3-2 mouse monoclonal antibody. This work was supported by research grants CA 56078, CA 80734 and CA 90878 from the National Cancer Institute, and research support from the Indiana Cancer Research Institute to AR Safa.

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Author notes

  1. Mojgan Rastegar

    Present address: McGill Cancer Center, 3655 Drummond Street, Montreal, H3G 1Y6, QC, Canada

  2. Besim Ogretmen

    Present address: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Strom Thurmond Building, 114 Doughty Street, Room 605, P.O. Box 250777, Charleston, 29425, SC, USA

Authors and Affiliations

  1. Department of Pharmacology and Toxicology, Indiana University, 1044 West Walnut R4-119, Indianapolis, 46202, Indiana, IN, USA

    Ching-Huang Wu, John Gordon, Mojgan Rastegar & Ahmad R Safa

  2. Indiana University Cancer Center, Indiana University, 1044 West Walnut R4-119, Indianapolis, 46202, Indiana, IN, USA

    Ching-Huang Wu, John Gordon, Mojgan Rastegar & Ahmad R Safa

  3. Hollings Cancer Center, Medical University of South Carolina, 86 Jonathan Lucas Street, Charleston, 29425, South Carolina, SC, USA

    Besim Ogretmen

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Correspondence to Ahmad R Safa.

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Wu, CH., Gordon, J., Rastegar, M. et al. Proteinase-3, a serine protease which mediates doxorubicin-induced apoptosis in the HL-60 leukemia cell line, is downregulated in its doxorubicin-resistant variant. Oncogene 21, 5160–5174 (2002). https://doi.org/10.1038/sj.onc.1205639

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