Research Papers:

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

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

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Oncotarget. 2018; 9:17104-17116. https://doi.org/10.18632/oncotarget.24936

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Angie B. Dull1, Deborah Wilsker1, Melinda Hollingshead2, Christina Mazcko3, Christina M. Annunziata4, Amy K. LeBlanc3, James H. Doroshow5, Robert J. Kinders1 and Ralph E. Parchment1

1Clinical Pharmacodynamic Biomarkers Program, Applied/Developmental Research Directorate, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA

2Biological Testing Branch, National Cancer Institute-Frederick, Frederick, Maryland, USA

3Comparative Oncology Program, National Cancer Institute, Bethesda, Maryland, USA

4Women’s Malignancies Branch, National Cancer Institute, Bethesda, Maryland, USA

5Division of Cancer Treatment and Diagnosis and Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA

Correspondence to:

Deborah Wilsker, email: [email protected]

Ralph E. Parchment, email: [email protected]

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

Received: January 19, 2018     Accepted: March 06, 2018     Published: March 30, 2018


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.

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