The predominant role of apoptosis in γH2AX formation induced by aneugens is useful for distinguishing aneugens from clastogens

Harada, Asako; Matsuzaki, Kaori; Takeiri, Akira; Mishima, Masayuki

doi:10.1016/j.mrgentox.2014.05.010

Cited by 22 publications

(15 citation statements)

References 35 publications

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“…37). It is well established that gH2AX is a biomarker of both DNA damage and apoptosis and that colocalization of gH2AX with cytoplasmic cleaved caspase-3-positive blebbing identifies the apoptotic cells (37)(38)(39). The gH2AX-positive cells seen in these samples also display visible chromatin condensation and fragmentation, consistent with them being apoptotic cells.…”

Section: Ddr Heterogeneity and The Role Of Cell Cyclementioning

confidence: 67%

Evaluation of Pharmacodynamic Responses to Cancer Therapeutic Agents Using DNA Damage Markers

Wilsker

Barrett

Dull

et al. 2019

Clinical Cancer Research

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Purpose: We sought to examine the pharmacodynamic activation of the DNA damage response (DDR) pathway in tumors following anticancer treatment for confirmation of target engagement. Experimental Design: We evaluated the time course and spatial activation of 3 protein biomarkers of DNA damage recognition and repair (gH2AX, pS343-Nbs1, and Rad51) simultaneously in a quantitative multiplex immunofluorescence assay (IFA) to assess DDR pathway activation in tumor tissues following exposure to DNA-damaging agents. Results: Because of inherent biological variability, baseline DDR biomarker levels were evaluated in a colorectal cancer microarray to establish clinically relevant thresholds for pharmacodynamic activation. Xenograft-bearing mice and clinical colorectal tumor biopsies obtained from subjects exposed to DNA-damaging therapeutic regimens demonstrated marked intratumor heterogeneity in the timing and extent of DDR biomarker activation due, in part, to the cell-cycle dependency of DNA damage biomarker expression. Conclusions: We have demonstrated the clinical utility of this DDR multiplex IFA in preclinical models and clinical specimens following exposure to multiple classes of cytotoxic agents, DNA repair protein inhibitors, and molecularly targeted agents, in both homologous recombinationproficient and-deficient contexts. Levels exceeding 4% nuclear area positive (NAP) gH2AX, 4% NAP pS343-Nbs1, and 5% cells with !5 Rad51 nuclear foci indicate a DDR activation response to treatment in human colorectal cancer tissue. Determination of effect-level cutoffs allows for robust interpretation of biomarkers with significant interpatient and intratumor heterogeneity; simultaneous assessment of biomarkers induced at different phases of the DDR guards against the risk of false negatives due to an ill-timed biopsy.

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Section: Ddr Heterogeneity and The Role Of Cell Cyclementioning

confidence: 67%

Evaluation of Pharmacodynamic Responses to Cancer Therapeutic Agents Using DNA Damage Markers

Wilsker

Barrett

Dull

et al. 2019

Clinical Cancer Research

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“…Harada et al (121) added caspase-3 staining in the in vitro gamma-H2AX assay with human lymphoblastoid TK6 cells to further classify gamma-H2AX-positive (gamma-H2AX+) cells into caspase-3positive (gamma-H2AX+/caspase-3+) for apoptosis and caspase-3-negative (gamma-H2AX+/caspase-3-) for DNA-damaged populations. The two clastogens, mitomycin C and etoposide, caused a significant increase of gamma H2AX+/caspase-3-cells but did not increase gamma-H2AX+/caspase-3+ cells significantly.…”

Section: Gamma-h2ax For Classification Of Clastogens and Aneugensmentioning

confidence: 99%

Chromosomal aberrations clastogens vs aneugens

Mishima¹

2017

Front Biosci

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Current anticancer therapy may be one of the most important exogenous sources of exposure to genotoxic agents in US, Japan, and Europe, where approximately 40-55 percent of the population is diagnosed with cancer at a certain point in their life. This review focuses on recent efforts to integrate a novel biomarker, gamma-H2AX, into anticancer drug screening to classify the mode of action (MoA) for genotoxic outcome into clastogenicity and aneugenicity, a distinction that has considerable impact on risk assessment and control strategy. The emerging biomarker gamma-H2AX is applicable to high throughput assay platforms and is therefore changing in vitro mammalian genotoxicity screening from traditional positive/negative selection to MoA elucidation. Because gamma-H2AX is not only a sensitive biomarker for DNA double strand break but is also induced by apoptosis, the key for successful screening is using additional biomarkers of caspase-3 and/or phosphorylated histone H3 to discriminate between relevant and irrelevant elevation of gamma-H2AX. Establishment of a standard methodology and a consensus threshold for its positive criteria will further support the application of gamma-H2AX to drug screening.

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“…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. However, 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 ]. Therefore, the observed γH2AX signal in clinical specimens could be indicative of apoptosis rather than DNA repair.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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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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The predominant role of apoptosis in γH2AX formation induced by aneugens is useful for distinguishing aneugens from clastogens

Cited by 22 publications

References 35 publications

Evaluation of Pharmacodynamic Responses to Cancer Therapeutic Agents Using DNA Damage Markers

Evaluation of Pharmacodynamic Responses to Cancer Therapeutic Agents Using DNA Damage Markers

Chromosomal aberrations clastogens vs aneugens

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

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