Increase, DNA strand breaks leads to Apoptosis

DNA strand breaks activate a series of DNA damage response (DDR) pathways that together determine whether damaged cells go through cell cycle arrest, repair, or cell death. When the levels of DNA damage exceed the DNA repair capacity, DDR signaling shifts the cellular response toward elimination of damaged cells by various forms of cell death. Thus, DNA strand breaks are potent inducers of programmed cell death (i.e., apoptosis) when damage is irreparable or sustained.

The empirical support for this KER is strong based on a large body of literature connecting DNA strand breaks and the induction of apoptosis. Both SSBs and DSBs are frequently associated with increased apoptosis in a dose-concordant manner in somatic and germ cells, as well as animal models (Roos and Kaina, 2006). This concordance has been observed following exposures to many DNA-damaging agents; select examples in different tissues and models are provided below.

Evidence in vivo (exposure)

Saha et al. (2014) demonstrated clear temporal and dose concordance between DSB formation and apoptosis in the embryonic mouse brain. Mice were exposed in utero to acute doses of 10, 25, 50, 100 or 200 mGy X-rays, and embryo brains were examined 1 hour or 6 hour later. The number of 53BP1 foci per cell was quantified as a marker of DSB induction, while TUNEL staining (Terminal deoxynucleotidyl UTP Nick End Labeling) was used to monitor apoptosis. Significant increases in 53BP1 foci formation (at 1 hour) and TUNEL+ apoptotic cells (at 6 hours) were observed starting at 10 mGy, indicating that the two KEs occur within the same dose range and that DSBs occur earlier than apoptosis. At 6 hours post-irradiation, while the number of 53BP1 foci at lower doses remained relatively unchanged, it was reduced at50 mGy and returned to the control level at 100 mGy, indicating efficient repair. Despite this apparent resolution of DNA damage, apoptosis continued to increase in a dose-dependent manner, suggesting that even low and transient DSBs are sufficient to induce apoptosis in this sensitive tissue (Saha et al., 2014).

Acetaminophen (200 mg/kg) induces DNA fragmentation and apoptosis in hepatocytes after intraperitoneal administration in mice (Bajt et al., 2011). Liver cells collected from exposed mice showed the release of histone-associated DNA fragments, mitochondrial pro-apoptotic proteins (apoptosis-inducing factor AIF, Smac, endonuclease G, and cytochrome c) into the cytosol at 6 hours post-treatment, accompanied by increased TUNEL+ staining at both 6 and 24 hours. This study also demonstrates the essentiality of mitochondrial pathways through the use of AIF-deficient mice. When treated with acetaminophen, these mice showed a marked reduction in presence of DNA strand breaks (TUNEL assay), % DNA fragmentation, and the expression of apoptotic markers compared to wild-type counterparts. This indicates that AIF is a necessary mediator of DNA fragmentation and apoptosis (Bajt et al., 2011). However, detection of DNA fragments does not allow discrimination between upstream DNA damage and downstream apoptotic DNA fragmentation (see Uncertainties and Inconsistencies). As only a single dose and timepoint were assessed for most markers, the data primarily support co-occurrence.

Evidence in vivo (genetic manipulation)

Using Nestin-Cre mice, Rodrigues et al. (2013) showed that deletion of Nibrin (Nbn, a key sensor of DSBs) and Atm kinase in neural stem/progenitor cells led to DSB accumulation (γH2AX+ cells, p53 stabilization) and concordantly enhanced apoptosis (TUNEL+ cells and cleaved caspase-3 staining) in specific regions of the developing brain (cerebellum, ganglionic eminences) and eye (lens) on embryonic days E15.5 and E17.5. The incidence of γH2AX+ cells (~6,000 to 10,000 cells/mm²) was consistently higher than that of TUNEL+ cells (~250 to 800 cells/mm²) and caspase-3+ cells (~200 to 800 cells/mm²) in the embryonic brains (Figure 1). A similar pattern was observed in eye lens and retina, supporting incidence concordance. Greater impairment of DDR signaling (Nbn/Atm^Nes-Cre double deletion) induced more DSBs and higher apoptosis than Nbn single deletion alone (Nbn^Nes-Cre) (Figure 1). This establishes a graded response-response relationship between DSB formation and apoptosis (Rodrigues et al., 2013). However, in the retina region, apoptosis induced by Nbn deletion was prevented by simultaneous inactivation of ATM, despite the presence of DNA damage. These findings provide direct evidence of essentiality, showing that key signaling pathways downstream of DNA strand breaks are required for the induction of subsequent apoptotic cell death. The observed differences across tissue suggest that these responses are context-dependent and may be mediated by tissue-specific pathways (Rodrigues et al., 2013).

Loss of Apc (adenomatous polyposis coli) in mouse liver tissue led to an early increase in DSBs, as indicated by elevated γH2AX and Rad51 staining at day 4, accompanied by upregulation of DNA damage checkpoint proteins p53 and p21 (Méniel et al., 2015). This was followed by a subsequent increase in apoptotic cells at day 6. Quantitative immunohistochemistry analysis revealed that the incidence of γH2AX-positive and Rad51-positive cells was consistently higher than that of caspase-3-positive apoptotic cells. At day 4, γH2AX-positive cells increased from 0.9% in WT to 26.2% in Apc-deficient mice and 67.1% in Apc/p53 double knockout mice, while Rad51 increased from 2% in WT to 9.9% and 20.7% after genetic manipulation (Figure 2). At the same timepoint, caspase-3-positive cells remained low (~0.36-1.34% across genotypes). A similar pattern was observed at day 6. Additional deletion of p53 further increased markers of DNA damage, indicating that p53 plays an essential role in regulating DSB levels following Apc loss. Together, these findings support incidence concordance and temporal sequence in which DSBs precede activation of checkpoint signaling and subsequent induction of p53-dependent apoptosis.

Xiao et al. (2023) established a myocardial ischemia/reperfusion mouse model to study the role of a long non-coding RNA (cardiac ischemia reperfusion associated Ku70 interacting lncRNA, or CIRKIL) in coronary artery disease. Overexpression of CIRKIL in mouse hearts and 100 μmol/L H₂O₂-treated mouse cardiomyocytes induced parallel increases in DSBs (γH2AX expression), p53 activation, and apoptotic markers, including the ratio of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins (Bax/Bcl-2), caspase-3 activity, and TUNEL+ cells. Conversely, knockdown of CIRKIL, as well as single knockout or double knockout of CIRKIL and its partner Ku70, alleviated these myocardial injury and reduced markers of both DNA strand breaks and apoptosis. The consistent bidirectional changes in these endpoints by multiple genetic interventions supports the role of the CIRKIL/Ku70 pathway in linking DNA strand breaks to apoptotic responses, and provides indirect evidence of essentiality (Xiao et al., 2023).

Together, the in vivo evidence provides strong empirical support for temporal concordance between strand breaks and apoptosis. Concurrent changes in both KEs were often observed at the same dose level, supporting qualitative dose concordance. A few studies support incidence concordance. In addition, the data highlight that the progression from DNA strand breaks to apoptosis is influenced by tissue-specific sensitivity and mechanisms, which should be considered when evaluating the applicability of this KER.

Evidence in vitro

Roos et al. (2004) provided important mechanistic evidence linking DNA alkylation, impaired repair, DNA strand breaks, and apoptosis in human lymphocytes. O6-methylguanine (O6-MeG) is a key DNA lesion associated with apoptosis, and its removal is mediated by O6-MeG-DNA methyltransferase (MGMT). To prevent repair of O6-MeG, cells were pre-treated with O6-benzylguanine to inactivate MGMT, followed by treatment with O6-MeG-generating agents, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) or an anticancer drug temozolomide. In MGMT-depleted proliferating lymphocytes, O6-MeG lesions were processed by mismatch repair during DNA replication, leading to the formation of DSBs, as demonstrated by neutral comet assay and γH2AX expression analysis. MNNG (10 or 25 μM) induced DSBs within 3 hours and the level peaked at 24 hours post-exposure. Apoptosis (measured by sub-G1 DNA content analysis or Annexin V/PI staining) was only observed after 24 hours (and persisted up to 96 hours) following MNNG treatment (at multiple concentrations, up to 20 μM), occurring later than DSB formation. Similar effects were induced by temozolomide. Apoptosis was associated with p53 stabilization and upregulation of the Fas receptor, and was significantly reduced (~61%) by treatment with an anti-Fas neutralizing antibody. In contrast, non-proliferating cells showed minimal apoptosis at lower MNNG concentrations (≤ 10 μM), indicating that DNA replication and mismatch repair is required in converting O6-MeG to DSBs. Furthermore, ionizing radiation (5 Gy), which directly induces DSBs, triggered apoptosis in both proliferating and non-proliferating lymphocytes. Together, these data suggest that O6-MeG is converted during DNA replication into DSBs that trigger apoptosis via p53- and Fas-dependent pathways. In addition, MGMT inactivation provides direct evidence of essentiality, as impaired DNA repair enhances the persistence of DNA strand breaks and contributes to apoptosis.

He et al. (2018) studied the effects of loperamide (an antidiarrheal agent) on DNA damage and apoptosis in leukemia cell lines and human primary leukemia cells. Cells exposed to 5, 10, 20 μM loperamide for 24 hours exhibited clear evidence for DNA strand breaks, which were detected by the alkaline comet assay (identifying SSBs and alkali-labile sites) and increased expression of γH2AX. In the same concentration range, a dose-dependent increase in the percentage of apoptotic cells was quantified by Annexin V/7-aminoactinomycin D+ staining and flow cytometry. The induction of apoptosis was further confirmed by increased protein expression of two apoptotic markers, cleaved-caspase-3 and cleaved poly (ADP‑ribose) polymerase (PARP), and decreased expression of an anti‑apoptosis protein Mcl‑1 at 10 μM and 20 μM. Activation of the ATM‑CHK2 pathway following 5‑20 µM loperamide treatment supported the induction of a DDR pathway in leukemia cells. As an example, the effects of loperamide on strand break formation and apoptosis in Molt-4 acute lymphocytic leukemia cells are shown in Figure 3. Similar effects were observed in Thp1 acute myelocytic leukemia cells and primary human leukemia cells (He et al., 2018).

Similarly, exposure to quercetin (40-80 μM) and the positive control paclitaxel (0.45 μM) for 48 hours induced simultaneous strand breaks and apoptosis in MGMT-positive human glioblastoma T98G cells (Wang et al., 2023). DNA strand breaks were assessed using the alkaline and neutral comet assays, and γH2AX staining, capturing both SSBs and DSBs. Apoptosis was assessed by the TUNEL assay, Annexin V-FITC and PI double staining, the ratio of Bax/Bcl-2, and the expression of apoptosis-related proteins, including cleaved caspase-9, cleaved caspase-3, and cleaved PARP. Quercetin (starting at 40 μM) and paclitaxel induced concentration-dependent increases in tail moment (tail length × % tail DNA) in the comet assays and γH2AX expression, indicating DSB formation. At the same concentrations and timepoint, both compounds significantly increased apoptotic cells, accompanied by a higher Bax/Bcl-2 ratio and elevated expression of apoptosis-related markers. Notably, quercetin (≥ 40 μM) also downregulated MGMT expression and activity, suggesting reduced MGMT-mediated DNA repair (Wang et al., 2023). These data support dose and temporalconcordance between DNA strand breaks and apoptosis; the concurrent downregulation of MGMT supports a role of insufficient DNA repair in the accumulation of DNA strand breaks and subsequent apoptosis.

Yauk et al. (2016) studied micronucleus induction and apoptosis in human lymphoblastoid TK6 cells following 4-hour exposures to six genotoxic chemicals: 2-aminoanthracene (0.75-2 μg/mL), acetaminophen (1-10 mM), cyclophosphamide (1-20 μM), dibenz[a,h]anthracene (1-280 μg/mL), dimethylnitrosamine (1-10 mM), furan (2-5 mM) (Figure 4). Micronucleus frequency serves as a marker of chromosomal damage resulting from unrepaired DNA lesions (including DSBs), but it is not a direct measure of DNA strand breaks. Consistent dose-dependent increases in both micronucleus frequency and the number of apoptotic cells were observed 20 hours post-exposure across all tested chemicals (Figure 4). In general, significant increases in apoptosis occurred at slightly lower concentrations than those required to induce micronucleus formation, and fewer cells exhibited chromosome damage than apoptosis, indicating a lack of incidence and only partial dose concordance. These observations likely reflect differences in assay sensitivity and biological progression, as micronucleus formation is a late-stage cytogenetic outcome that requires progression through mitosis. Therefore, these differences do not contradict the overall relationship. As both endpoints were measured at the same time, these data support temporal concordance of this KER.

Liu et al. (2014) showed that inhibition of the PI3K/AKT pathway (a regulator of DDR) by BKM120 led to DNA damage and increased apoptosis in human and murine hepatocellular carcinoma cell lines (Huh7 and BNL). Cells pretreated with 1 μM BKM120 for 1 hour were irradiated (10 Gy for Huh7 cells and 5 Gy for BNL cells). Radiation induced DSB formation (assessed by γH2AX staining) within 30 minutes and the presence of the PI3K inhibitor led to prolonged foci retention (≥ 8 hours in Huh7 cells and ≥ 4 hours in BNL cells). In parallel, PI3K inhibition increased radiation-induced apoptosis, as indicated by elevated cleaved caspase-3 expression and increased Annexin V-positive cells at 24 hours. Co-treatment with rapamycin, an mTOR inhibitor downstream of PI3K, further enhanced both DSB persistence and apoptosis (Liu et al., 2014). These findings demonstrate strong temporal concordance, with DSBs occurring earlier than apoptotic responses; parallel increases were observed across experimental conditions. Although dose-response data are not available, modulation of DNA damage persistence through PI3K/AKT and downstream mTOR inhibition provides indirect essentiality support for the link between DNA strand breaks and apoptosis.

Additional empirical evidence from Chen et al. (2022) supports this KER in human nasopharyngeal carcinoma cells. Two cell lines, SUNE1 and HONE1, were transfected to overexpress ubiquitin-specific protease 44 (USP44). USP44 regulates ubiquitin signaling at DSB sites, thereby modulating DDR and repair processes. Following irradiation (6 Gy), both cell lines exhibited rapid induction of DNA strand breaks, as shown by significant increases in tail moment in the neutral comet assay and higher γH2AX foci numbers within 30 minutes to 4 hours post-exposure. Minimal or no DNA damage was observed at 24 hours, indicating efficient repair at later timepoints. In the same system, USP44 overexpression promoted radiation-induced apoptosis, demonstrated by Annexin V/PI staining at 24 hours in vitro and increased caspase-3 staining in subcutaneous xenograft tumors in mice (Chen et al., 2022). The alignment between early DSB formation and subsequent apoptotic responses supports strong temporal concordance; concurrent increases in both KEs following USP44 overexpression provide indirect essentiality evidence for this KER.

CDKN2AIP is a cell cycle-associated protein that has been implicated in the regulation of DNA damage repair, p53-dependent apoptosis, and spermatogenesis. An in vitro study by Cao et al. (2022) using Cdkn2aip-knockdown TM4 mouse testicular Sertoli cells exposed to γ-irradiation (5 Gy) showed impaired DSB repair, evidenced by persistent γH2AX and 53BP1 foci formation at 2h, 12h, and 24h, together with a significant increase in apoptosis measured by flow cytometry and TUNEL at 24h (Cao et al., 2022). This study provides evidence for temporal concordance and essentiality.

Overall, across multiple somatic cell types, radiation-induced, chemically or genetically induced DNA strand breaks are consistently associated with an increase in apoptosis. These studies demonstrate strong temporal concordance, with DNA strand breaks occurring early or concurrently with apoptotic responses. Evidence for dose concordance is supported in several studies but dose-response data are not always available. Evidence for incidence concordance is limited in vitro. The consistent direction of responses across experimental conditions provide strong empirical support for the relationship between these two KEs in somatic cells.

Evidence in germ cells

A study by Hamer et al., (2003) provided evidence linking DSBs and apoptotic responses in male germ cells. They first demonstrated the presence of basal γH2AX in testes of wild-type FvB/NAU mice. Following a single ionizing radiation (4 Gy), γH2AX foci were rapidly (within 3 hours) induced in spermatogonia, spermatocytes, and round spermatids. In spermatogonia, γH2AX and p53 colocalized in the nucleus and their interaction was enhanced following irradiation. These findings support co-occurrence of DSBs and p53-dependent apoptosis in spermatogonia.

Habas et al. reported the effects of known mutagens on testicular germ cells isolated from 10-12-week-old NMRI mice (National Medical Research Institute) or male Sprague-Dawley rats. Isolated testicular germ cells (spermatogonia, spermatocytes and spermatids) were exposed for 1 hour to one of seven genotoxic compounds at 50, 500, or 1000 μM, and were immediately subjected to alkaline comet or TUNEL analyses. The compounds included doxorubicin (Dox), which induces DNA strand breaks via topoisomerase II inhibition (Habas et al., 2017), and a panel of mutagens targeting germ cells at specific stages: alkylating agents N-ethyl-N-nitrosourea (ENU) and N-ethyl-N-nitrosourea (MNU), which target pre-meiotic spermatogonia; 6-mercaptopurine (6-MP) and 5-bromo-20-deoxy-uridine (5-BrdU), which produce the greatest responses in early meiotic spermatocytes; and methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS) that primarily target post-meiotic germ cells (Habas et al., 2016). Representative results (Figure 5) show highly consistent dose-dependent increases in both % tail DNA in the alkaline comet assay and the percentage of TUNEL+ apoptotic cells across male germ cells at all stages, with both effects being statistically significant at the same exposure concentrations (Habas et al., 2016, 2017). These data support dose and temporal concordance while extending the applicability of this KER to male germ cells.

Cao et al. (2022) demonstrated that loss of CDKN2AIP leads to a concurrent increase in DNA strand breaks and apoptosis in male germ cells. CDKN2AIP preferentially expresses in spermatocyte and spermatids and acts as a central regulator of DSBs repair. Analyses of Cdkn2aip-/- male C57BL/6 mice revealed a significant accumulation of DSBs (γH2AX analyses) in pachytene spermatocytes from postnatal day P35 testes, along with a higher incidence of TUNEL foci per cells in the seminiferous tubules (Cao et al., 2022). These data indicate a clear association between increased DSBs and male germ cell apoptosis, and provide evidence for the essential role of DNA damage repair in preventing DSB accumulation and subsequent apoptotic cell death.

Suh et al. (2006) examined oocytes from Balb/c mice exposed to γ-irradiation (0, 0.1, 0.45 Gy) on postnatal day 5. At 24 hours post-exposure, on average, oocytes exposed to 0.1 Gy had an average of three γH2AX-marked DSBs with minimal apoptosis; oocytes receiving 0.45 Gy had about ten DSBs and underwent apoptosis within 24 hours, as indicated by condensed chromatin and positive TUNEL staining (Suh et al., 2006). Consistent with early DNA damage induction, Stringer et al. (2020) reported dose- and time-dependent increases in nuclear localization of phosphorylated ATM in mouse oocytes as early as 30 minutes following irradiation (0.2, 0.45, 7 Gy), indicating activation of DNA damage responses. The presence of DSBs was evidenced by the formation of γH2AX foci at all doses. Apoptosis was indirectly monitored by follicle survival at 24 hours and oocyte retrieval rate. At the same timepoints and dose range, Rad51 (a marker of homologous recombination) localized to DNA damage sites in over 90% of oocytes in primordial follicles, whereas DNA-dependent protein kinase catalytic subunit (DNA-PKcs, a marker of non-homologous end joining) was mostly present in granulosa cells; this difference suggests cell type-specific utilization of repair pathways. Importantly, intervention studies in this system provide indirect evidence of essentiality. In Tap63 knockout mice (deficient in DNA damage-induced apoptotic signaling), primordial follicles were preserved despite the presence of DNA damage. Conversely, pharmacological inhibition of Rad51 increased persistence of unrepaired DNA damage and apoptosis, suggesting that efficient DNA repair mitigated the progression from DNA strand breaks to cell death.

Experimental evidence from isolated germ cells demonstrates that DNA strand breaks are closely associated with increased apoptosis across multiple stages of spermatogenesis and in oocytes. These findings support the applicability of this KER to germ cells and reinforce dose and temporal concordance.

Although this KER is widely accepted based on the qualitative evidence linking the presence of strand breaks to apoptosis, the quantitative understanding of this KER is limited, in part due to the sensitivity and specificity of available test systems and assays. Commonly used metrics of DNA strand breaks, such as the comet assay tail moment, are influenced by multiple variables including DNA repair kinetics, electrophoresis conditions, and cell type, and do not linearly reflect the absolute number of DNA breaks. Lab-to-lab variability and differences in detection thresholds across DNA damage and apoptosis assays (e.g., γH2AX, TUNEL, cleaved caspase-3) further constrain quantitative extrapolation.

Despite these limitations, some studies allow relative comparisons between upstream and downstream KEs, indicating that the incidence of DNA strand breaks is generally higher than that of apoptosis (Méniel et al., 2015), but the level of incidence varies across tissues. For example, Rodrigues et al., (2013) reported ~6,000 to 10,000 γH2AX+ cells/mm², ~250 to 800 TUNEL+ cells/mm², and ~200 to 800 caspase-3-positive cells/mm² in the cerebellum of E15.5-17.5 Nbn/Atm^-/- mice; in contrast, lower counts were observed in the retina at the developmental stage, with only ~1,500 to 2,800 γH2AX+ cells/mm² and ~20 to 60 TUNEL+ cells/mm² counted (Figure 1) (Rodrigues et al., 2013). It remains challenging to define a precise threshold of DNA damage required to trigger apoptosis.  

In addition, the quantitative relationship is likely influenced by different sensitivity across biological systems. For example, Saha et al. (2014) estimated that as few as one to two DSBs induced by X-ray radiation are sufficient to activate apoptosis in the embryonic mouse brain. Notably, embryonic neuronal stem and early progenitor cells are among the most radiosensitive cells; this sensitivity allows researcher to monitor both initial damage and subsequent apoptosis. Some studies propose that oocytes exhibit an exceptionally low apoptotic threshold, such that even a single or a few irreparable DNA breaks may trigger cell death (Suh et al., 2006; Stringer et al., 2020). It is unclear whether a certain number of strand breaks is required to trigger apoptosis in less-sensitive or more complex systems. The quantitative understanding of this KER may not be generalized across cell types, tissues, or developmental stages.

This KER applies to eukaryotic cells exposed to genotoxic stressors that induce persistent or unrepaired DNA strand breaks. However, the applicability of this KER is context-dependent and influenced by cell type, developmental stage, DNA repair capacity, and DDR signaling status.

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