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.
Highlights
The histone protein H2AX is phosphorylated on serine 139 to form S139-phosphorylated histone H2AX (γH2AX) at sites of DNA doublestrand breaks (DSBs), and this phosphorylation is required for the recruitment of DNA repair factors after DNA damage
The γH2AX immunofluorescence assay (IFA) we developed for analysis of clinical specimens has been widely used across numerous NCI-sponsored clinical trials to reliably detect double strand break (DSB) in tumor cells examined in clinical specimens
Caspase-mediated cleavage of type I keratins during apoptosis is known to lead to formation of such cleaved caspase-3 (CC3)-containing puncta independent of γH2AX [4, 17, 18], and we found that these CC3 puncta were associated with the membrane blebbing that is characteristic of apoptotic cells (Supplementary Figure 1)
Summary
The histone protein H2AX is phosphorylated on serine 139 to form γH2AX at sites of DNA doublestrand breaks (DSBs), and this phosphorylation is required for the recruitment of DNA repair factors after DNA damage. Our lab and others have demonstrated the appearance of γH2AX after treatment with genotoxic agents by immunofluorescence assay using formalinfixed, paraffin-embedded tumor tissue [1,2,3]. The γH2AX immunofluorescence assay (IFA) we developed for analysis of clinical specimens has been widely used across numerous NCI-sponsored clinical trials to reliably detect DSBs in tumor cells examined in clinical specimens. While the function of γH2AX was originally believed to be associated primarily with DNA repair, its role as a marker in DNA ladder formation during apoptosis has been well-established [4, 5]. We sought to resolve this ambiguity by developing an immunofluorescence microscopy assay to identify and enumerate γH2AXpositive cells undergoing apoptosis
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