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Dull AB, Wilsker D, Hollingshead M, et al. Development of a quantitative pharmacodynamic assay for apoptosis in fixed tumor tissue and its application in distinguishing cytotoxic drug-induced DNA double strand breaks from DNA double strand breaks associated with apoptosis. Oncotarget. 2018;9(24):17104-17116doi: 10.18632/oncotarget.24936.
Dull, A. B., Wilsker, D., Hollingshead, M., Mazcko, C., Annunziata, C. M., LeBlanc, A. K., Doroshow, J. H., Kinders, R. J., & Parchment, R. E. (2018). Development of a quantitative pharmacodynamic assay for apoptosis in fixed tumor tissue and its application in distinguishing cytotoxic drug-induced DNA double strand breaks from DNA double strand breaks associated with apoptosis. Oncotarget, 9(24), 17104-17116. https://doi.org/10.18632/oncotarget.24936
Dull, Angie B, et al. "Development of a quantitative pharmacodynamic assay for apoptosis in fixed tumor tissue and its application in distinguishing cytotoxic drug-induced DNA double strand breaks from DNA double strand breaks associated with apoptosis." Oncotarget vol. 9,24 (2018): 17104-17116. doi: https://doi.org/10.18632/oncotarget.24936
Dull AB, Wilsker D, Hollingshead M, Mazcko C, Annunziata CM, LeBlanc AK, Doroshow JH, Kinders RJ, Parchment RE. Development of a quantitative pharmacodynamic assay for apoptosis in fixed tumor tissue and its application in distinguishing cytotoxic drug-induced DNA double strand breaks from DNA double strand breaks associated with apoptosis. Oncotarget. 2018 Mar 30;9(24):17104-17116. doi: 10.18632/oncotarget.24936. eCollection 2018 Mar 30. PMID: 29682208; PMCID: PMC5908309.
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Oncotarget. 2018 Mar 30;9(24):17104-17116. doi: 10.18632/oncotarget.24936. eCollection 2018 Mar 30.

Development of a quantitative pharmacodynamic assay for apoptosis in fixed tumor tissue and its application in distinguishing cytotoxic drug-induced DNA double strand breaks from DNA double strand breaks associated with apoptosis.

Oncotarget

Angie B Dull, Deborah Wilsker, Melinda Hollingshead, Christina Mazcko, Christina M Annunziata, Amy K LeBlanc, James H Doroshow, Robert J Kinders, Ralph E Parchment

Affiliations

  1. Clinical Pharmacodynamic Biomarkers Program, Applied/Developmental Research Directorate, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.
  2. Biological Testing Branch, National Cancer Institute-Frederick, Frederick, Maryland, USA.
  3. Comparative Oncology Program, National Cancer Institute, Bethesda, Maryland, USA.
  4. Women's Malignancies Branch, National Cancer Institute, Bethesda, Maryland, USA.
  5. Division of Cancer Treatment and Diagnosis and Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.

PMID: 29682208 PMCID: PMC5908309 DOI: 10.18632/oncotarget.24936

Abstract

DNA double strand breaks (DSBs) induced by cancer therapeutic agents can lead to DNA damage repair or persistent DNA damage, which can induce apoptotic cell death; however, apoptosis also induces DSBs independent of genotoxic insult. γH2AX is an established biomarker for DSBs but cannot distinguish between these mechanisms. Activated cleaved caspase-3 (CC3) promotes apoptosis by enhancing nuclear condensation, DNA fragmentation, and plasma membrane blebbing. Here, we describe an immunofluorescence assay that distinguishes between apoptosis and drug-induced DSBs by measuring coexpression of γH2AX and membrane blebbing-associated CC3 to indicate apoptosis, and γH2AX in the absence of CC3 blebbing to indicate drug-induced DNA damage. These markers were examined in xenograft models following treatment with topotecan, cisplatin, or birinapant. A topotecan regimen conferring tumor regression induced tumor cell DSBs resulting from both apoptosis and direct DNA damage. In contrast, a cisplatin regimen yielding tumor growth delay, but not regression, resulted in tumor cell DSBs due solely to direct DNA damage. MDA-MB-231 xenografts exposed to birinapant, which promotes apoptosis but does not directly induce DSBs, exhibited dose-dependent increases in colocalized γH2AX/CC3 blebbing in tumor cells. Clinical feasibility was established using formalin-fixed, paraffin-embedded biopsies from a canine cancer clinical trial; γH2AX/CC3 colocalization analysis revealed apoptosis induction by two novel indenoisoquinoline topoisomerase I inhibitors, which was consistent with pathologist-assessed apoptosis and reduction of tumor volume. This assay is ready for use in clinical trials to elucidate the mechanism of action of investigational agents and combination regimens intended to inflict DNA damage, apoptotic cell death, or both.

Keywords: DNA double strand breaks; apoptosis; immunofluorescence; multiplex; pharmacodynamic assay

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

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