DNA damage-dependent mechanisms of ionizing radiation-induced cellular senescence

Introduction

Aging represents a complex biological phenomenon fundamentally characterized by cellular senescence (Muller, 2009), driven by intricate molecular mechanisms. Cellular senescence refers to a stable, non-proliferative state marked by cell cycle arrest, diminished cell function, resistance to cell death, upregulated expression of the cell cycle inhibitory gene p16^INK4a, as well as a series of biological changes within the cellular environment. Cellular senescence manifests in two main forms. The first is replicative senescence (RS), also known as the Hayflick limit, which is caused by progressive telomere shortening with each cell division. When critical telomere length is reached, cells undergo permanent cell cycle arrest (Hayflick & Moorhead, 1961). The second type is stress-induced premature senescence (SIPS), which is triggered by external stressors and internal cellular damage, such as oxidative stress, DNA damage, inflammatory responses, ionizing radiation (IR), or exposure to certain chemical toxins. Leonard Hayflick first described the phenomenon of cellular senescence, observing that normal human embryonic cells divide approximately 40–60 times before entering senescence. This phenomenon, known as the ‘Hayflick limit’, suggests that the natural human lifespan is capped at around 120 years (Hayflick, 1965). This discovery established the foundational concept of cellular senescence and provided a critical experimental model for subsequent aging research. Cellular senescence can also be triggered by both intrinsic and extrinsic factors. Among these, IR is a potent extrinsic inducer of cellular aging, primarily through three interconnected mechanisms: direct induction of DNA damage, activation of cell cycle checkpoint, and amplification of oxidative stress. Together, these insults compromise cellular homeostasis and drive the transition to a senescent state, a process termed SIPS that phenotypically resembles RS (Suzuki & Boothman, 2008). At the molecular level, IR inflicts direct genomic damage in the form of base damage, single- and double-strand DNA breaks, which are repaired by pathways such as base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR), and non-homologous end joining (NHEJ). The cell cycle is tightly regulated by checkpoints (G1/S, intra-S, G2/M) controlled by p53-p21 signaling, which pause the cell cycle to allow repair. While low-doses of radiation typically allow for efficient DNA repair through endogenous mechanisms, higher doses often generate complex, persistent lesions that can evade repair for extended periods. The accumulation of such irreparable DNA damage serves as a critical trigger for senescence initiation (Rodier et al., 2011). Therefore, this review focuses specifically on the DNA damage-dependent pathways underlying IR-induced cellular senescence, particularly in scenarios where DNA repair capacity is overwhelmed or compromised.

The target audience of this article primarily encompasses cell biologists with a keen interest in the mechanisms of cellular senescence, DNA damage response, and cell cycle regulation, as well as molecular biologists who are engaged in the study of gene expression, signaling pathways, and protein interactions. Additionally, students and educators in the relevant fields may also use this article as an educational resource to learn about the latest research into IR-induced cellular senescence.

Survey Methodology

The PubMed database was utilized to search for relevant literature using keywords such as “Ionizing Radiation,” “Cellular Senescence,” “Senescence,” “Aging,” “DNA Damage,” “Cancer,” “DDR,” “NHEJ,” “ROS,” “SASP,” “p53,” “SA-β-galactosidase,” and “p21.” Additionally, some of the data utilized in this article were also sourced from Web of Science and Google Scholar. The literature citations aim to provide comprehensive and impartial coverage of the topic. The search criteria encompass a time span of the past 5 to 10 years, and even extend back to the seminal research articles.

Characteristics of Cellular Senescence

The senescent phenotype can be distinguished morphologically by cellular hypertrophy (increased size and flattening), nuclear abnormalities (enlargement or multinucleation), cytoplasmic alterations (enhanced granularity and vacuolization), and abnormal organelle morphology, particularly lysosomal expansion. Senescent cells exhibit four key hallmarks: (1) permanent cell-cycle arrest mediated by p53-p21 and p16-Rb pathways, (2) a pro-inflammatory senescence-associated secretory phenotype (SASP) that remodels the tissue microenvironment, (3) accumulation of macromolecular damage (e.g., DNA breaks, oxidized proteins, and lipofuscin), and (4) altered metabolism, including mitochondrial dysfunction and enhanced lysosomal activity (Gorgoulis et al., 2019; Humphreys, ElGhazaly & Frisan, 2020).

The irreversible cell-cycle arrest in senescence is primarily mediated by two interconnected tumor suppressor pathways. The p53-p21 axis responds to DNA damage and oxidative stress, where stabilized p53 transcriptionally activates the cyclin-dependent kinase (CDK) inhibitor p21, blocking cyclin-CDK complexes to prevent Rb phosphorylation and E2F-mediated S-phase entry. In parallel, the p16-Rb pathway reinforces arrest through p16-mediated inhibition of CDK4/6, maintaining Rb in its active, hypophosphorylated state to permanently suppress E2F target genes. This arrest is stabilized by epigenetic silencing and sustained by SASP factors, creating a failsafe mechanism against proliferation. Together, these pathways ensure senescence irreversibility through layered transcriptional and chromatin-level repression of cell-cycle progression (Sharpless & Sherr, 2015).

The SASP is a complex and dynamic collection of secreted factors produced by senescent cells. This includes a wide range of molecules, such as pro-inflammatory cytokines (e.g., IL-1α, IL-1β, IL-6, IL-8), chemokines (e.g., CCL2, CXCL1, CXCL10), growth factors (e.g., TGF-β, HGF), matrix metalloproteinases (e.g., MMP1, MMP3), lipids (e.g., prostaglandins), and extracellular vesicles (e.g., exosomes containing microRNAs). In a study by Jeon et al. (2023), it was observed that physiological aging is transferred from old mice to young mice following blood exchange, and it was pointed out that some factors are due to SASP-induced secondary senescence. The SASP is regulated by various signaling pathways, including the DNA damage response (DDR), NF-κB, p38 MAPK, mTOR, and cGAS–STING pathways, as well as epigenetic changes. Its composition and strength vary depending on the duration of senescence, the origin of the pro-senescence stimulus, and the cell type. The SASP plays a crucial role in mediating the non-cell-autonomous effects of senescent cells, influencing tissue microenvironments, immune responses, and contributing to both beneficial and detrimental biological functions in different physiological and pathological contexts (Wang et al., 2024a).

Senescent cells exhibit widespread macromolecular damage, encompassing a variety of molecular alterations. A defining feature is DNA damage, including persistent lesions such as double-strand breaks (DSBs), oxidative modifications, and telomere dysfunction. These lesions activate the DDR, triggering cell cycle arrest via key regulators such as ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3 related (ATR), and p53. Proteins in senescent cells also frequently undergo oxidative modifications—such as carbonylation and nitration—that impair their structure and function. Similarly, lipid peroxidation generates reactive aldehydes like 4-hydroxy-2-nonenal (4-HNE), which further modify proteins and lipids, exacerbating cellular dysfunction. A prominent consequence of this oxidative damage is the accumulation of lipofuscin, an autofluorescent, undegradable pigment that builds up in lysosomes. Composed of cross-linked peroxidation byproducts (e.g., malondialdehyde and 4-HNE bound to proteins), lipofuscin forms insoluble aggregates that resist lysosomal degradation. This reflects a decline in cellular clearance mechanisms and serves as a key biomarker of senescence and aging (Nousis, Kanavaros & Barbouti, 2023).

Cellular senescence is accompanied by substantial metabolic shifts, primarily manifested through changes in mitochondrial and lysosomal functions. The mitochondria of senescent cells often show decreased membrane potential, increased production of reactive oxygen species (ROS), and reduced efficiency in ATP synthesis. It has been found that the pluripotency transcription factor Nanog homeobox (NANOG) can rejuvenate dysfunctional mitochondria in senescent cells. Proline supplementation has been shown to rescue mitochondrial respiratory dysfunction in senescent mesenchymal stem cells by activating mitophagy and restoring metabolic homeostasis, thereby reducing senescence markers (Choudhury et al., 2024). Enhanced lysosomal function in senescent cells is marked by elevated levels of lysosomal enzymes, greater lysosomal mass, and enhanced autophagic flux. These factors contribute to the accumulation of undegraded material, such as lipofuscin, and the secretion of SASP. These lysosomal changes, including the elevated activity of the enzyme senescence-associated β-galactosidase (SA-β-gal) and the accumulation of oxidized material, have been particularly exploited as senescence biomarkers (Debacq-Chainiaux et al., 2009).

The presence or absence of senescent cells can have harmful effects depending on the context. For example, their accumulation in aged tissues exacerbates chronic inflammation and fibrosis, while their removal in developing organs (e.g., lungs) or during regeneration (e.g., liver) disrupts normal function. In addition, senescent cells were identified in resected glioblastoma multiforme (GBM) tissues from patients and in mouse GBM models, discovering that partially removing p16^INK4a-positive malignant senescent cells modulates the tumor microenvironment and improves survival outcomes in GBM mice (Salam et al., 2023). The definition of senescence remains controversial due to the lack of universal biomarkers—while p16 and SASP are commonly used, their expression varies across cell types. Additionally, senescence exhibits dual roles, acting beneficially in tumor suppression and wound healing but detrimentally in chronic diseases. This functional ambiguity, combined with inconsistent results from animal models, challenges the classification of senescence as purely pathological or physiological. Ultimately, the dynamic nature of senescent cells complicates their therapeutic targeting and necessitates context-specific definitions (López-Otín et al., 2023; De Magalhães, 2024).

DNA Damage Is an Important Indicator of Cellular Senescence

Cellular senescence is a state of irreversible cell cycle arrest that occurs in response to various stressors, including DNA damage. DNA damage arises constantly due to endogenous and exogenous factors. Under physiological conditions, endogenous instability of DNA leads to spontaneous lesions, such as the hydrolytic cleavage of glycosidic bonds, the deamination of bases and the formation of oxidative adducts. It is estimated that there are 10,000 to 100,000 lesions per cell per day (Yousefzadeh et al., 2021). Exogenous sources, including IR and environmental mutagens, further exacerbate this damage. To counteract these threats, cells rely on highly conserved DNA repair pathways. Mammalian cells employ four key repair mechanisms: BER for oxidative lesions, NER for bulky adducts, mismatch repair (MMR) for replication errors, and DSB repair via error-prone NHEJ, which is mediated by Ku70/80, or precise HR, which requires the MRE11-RAD50-NBS1 (MRN) complex (Huang & Zhou, 2021).

The accumulation of DNA damage, particularly complex and clustered lesions, is a critical driver of senescence, acting as both a cause and a consequence of senescence (Pustovalova et al., 2025). The reviewed literature highlights the pivotal role of DNA damage in inducing senescence, especially when the damage is severe or poorly repaired, as is often the case with high-linear energy transfer (LET) radiation (Mavragani et al., 2019). Unlike isolated lesions, clustered DNA damage—such as closely spaced DSBs, single-strand breaks (SSBs), and base lesions, poses a great challenge to cellular repair system. When cells fail to adequately resolve such damage, persistent DNA lesions activate the DDR pathway, leading to cell cycle arrest and senescence. For instance, studies have shown that high-LET radiation, such as carbon ions or alpha particles, generates highly clustered DNA lesions that are repaired more slowly or remain unrepaired, resulting in prolonged DDR activation and eventual senescence (Van De Kamp et al., 2021). This is supported by the increased expression of senescence markers, such as β-galactosidase, and the formation of senescence-associated heterochromatin foci (SAHF) in irradiated cells (Helm et al., 2016).

Evidence from progeroid syndromes, such as Werner syndrome and Hutchinson-Gilford progeria syndrome, highlights the consequences of defective DNA repair and accelerated senescence. These conditions are caused by mutations in genes involved in DNA maintenance (e.g., WRN and LMNA), resulting in patients exhibiting age-related pathologies at an early age (Kudlow, Kennedy & Monnat, 2007) . Similarly, studies in mouse models with DNA repair deficiencies, such as Ercc1 mutants, demonstrate increased DNA damage, senescence, and aging phenotypes that mirror natural aging processes (Vander Linden et al., 2024). These findings reinforce the idea that DNA damage is a critical indicator of cellular senescence and organismal aging.

Effects of Ir-Induced Dna Damage on Cellular Senescence

IR refers to radiation with sufficient energy to release electrons from atoms or molecules, causing them to become electrically charged. Such radiation can be electromagnetic waves or particle streams, including alpha rays, beta rays, X-rays, gamma rays, and neutron radiation (Fiorentino et al., 2009). IR causes cell senescence mainly by inducing DNA damage, halting cell cycle progression, increasing SASP secretion, increasing oxidative stress, and shortening telomeres (Fig. 1). However, Soroko et al. (2024) found that low-dose-rate (LDR) radiation significantly induces cellular senescence, manifested as an increase in SA-β-gal stain-positive cells, and this effect was dose-dependent. High-dose-rate (HDR) radiation did not significantly induce cellular senescence, which may be related to the increased apoptosis it causes. Aboussekhra et al. (2023) found that different doses of IR inhibit the activity of breast cancer-associated fibroblasts and their adjacent fibroblasts. Importantly, β-gal staining of breast stromal fibroblast cells was significantly increased after high-dose X-ray (50 Gy) treatment, demonstrating that high doses of IR induce senescence. IR can induce severe DNA damage, often leading to apoptosis and cellular senescence in vitro and in vivo, which is associated with delayed repair of irradiated tissues (Wang et al., 2019). IR induces cellular senescence and significantly impacts the expression of the SASP. Chronic DNA damage is also known to induce a high secretory state of SASP (Wang et al., 2024a). For example, in a study of human esophageal squamous cell carcinoma, it was found that radiotherapy can induce senescence in cancer cells and stimulate nearby cells through paracrine secretion of pro-inflammatory factors (Zhang et al., 2023). In studies of glioblastoma, Jeon et al. (2023) discovered that IR-induced tissue factor (F3) was strongly induced in senescent glioblastoma (GBM) cells, and F3 coordinates the remodeling of the oncogenic tumor microenvironment (TME) by activating tumor-autonomous signaling and the extrinsic coagulation pathway. In an investigation of the effects of IR on the induction of senescence in annular fibroblast (AF) cells, AF cells were exposed to 10-15 Gy of IR for five days. This exposure resulted in near-complete senescence, as evidenced by increased SA-β-gal staining and a significant elevation in SASP expression (Zhong et al., 2022). Isermann, Mann & Rübe (2020) found that the histone variant H2A.J is closely associated with IR-induced cellular senescence and the DDR. In H2A.J-deficient mice, there was a notable increase in SASP factor secretion in the skin. H2A.J regulates the expression of SASP-related genes through its influence on chromatin structure, thereby playing a critical role in modulating the SASP during IR-induced senescence. Certain SASP factors accelerate cell senescence, with IL-6 and IL-8 being the most extensively studied. IL-6 and IL-8, two pro-inflammatory cytokines, are typically expressed at high levels in senescent cells. Elevated IL-6 and IL-8 levels play a crucial role in reinforcing senescence and promoting inflammation (Wang et al., 2024b).

IR generates a substantial amount of ROS, which can cause oxidative damage to cellular macromolecules including DNA, proteins, and lipids (Terman, 2001). Moreover, increased production of ROS within the cell is closely associated with the accumulation of dysfunctional mitochondria (Guilbaud, Sarosiek & Galluzzi, 2024). Radiation induces cellular senescence and the accumulation of dysfunctional mitochondria in salivary gland organoids, leading to disrupted mitochondrial dynamics, reduced mitochondrial DNA copy number, and increased ROS production (Cinat et al., 2024). Research indicates that nicotinamide riboside (NR) can significantly alleviate intestinal aging induced by IR, with mechanisms including reducing oxidative damage, restoring normal function of intestinal stem cells, regulating disruption of the gut symbiotic ecosystem, and resolving metabolic abnormalities (Yue et al., 2024). In addition, Zhong et al. (2022) demonstrated that IR induces cellular senescence in annulus fibrosus cells through the oxidative stress pathway and activates MMP-mediated matrix degradation pathways, leading to degeneration and dysfunction of the intervertebral disc tissue. Therefore, an increase in ROS or a decrease in antioxidant responses will accelerate cellular senescence.

Here, we propose that radiation-induced DNA damage triggers cellular senescence through three distinct yet interconnected mechanisms. First, radiation-mediated DNA damage can induce cellular growth arrest by activating cell cycle checkpoints (Campisi, 2013). This protective response allows cells to temporarily halt proliferation upon detecting DNA lesions, providing time for DNA repair while preventing the transmission of damaged genetic material during replication (Bekker-Jensen & Mailand, 2010). Second, IR generates severe DNA lesions, including DSBs or DNA crosslinks, which frequently exceed the cell’s intrinsic repair capacity (Rodier et al., 2009). The accumulation of unrepaired DNA damage leads to persistent genomic instability, ultimately compromising cellular viability. Third, IR can inflict damage to telomeric DNA, thereby accelerating the senescence process. Table 1 summarizes the key evidence from recent studies that supports the three proposed mechanisms by which radiation-induced DNA damage triggers cellular senescence. Cellular senescence represents the terminal outcome of these cascading events. Prolonged exposure to DNA damage and diminished repair capacity eventually leads to cellular functional decline and the onset of aging.

Conclusion

IR, a potent genotoxic stressor, induces diverse DNA lesions, including base damage, SSBs, and DSBs, with clustered damage—particularly from high-LET radiation—posing a significant challenge to repair systems. When repair capacity is overwhelmed, persistent DNA damage triggers the DDR, activating cell cycle checkpoints via the ATM/ATR-CHK1/CHK2 axis and reinforcing senescence through the p53-p21 and p16-Rb pathways. These cascades lead to irreversible cell cycle arrest, a defining feature of senescence, while simultaneously promoting the SASP secretion. The SASP not only perpetuates senescence but also fosters a pro-inflammatory microenvironment. Additionally, IR-induced telomere dysfunction mimics replicative senescence by recruiting DDR proteins to chromosomal ends. Mitochondrial dysfunction and ROS production further amplify DNA damage, creating a vicious cycle.

Although persistent DSBs typically trigger apoptosis or senescence, the fate of irradiated cells depends on their molecular context. The role of IR-induced senescence in cancer treatment is a double-edged sword. On one hand, IR-induced senescence directly inhibits tumor proliferation by permanently arresting the cell cycle (dependent on the p53-p21 and p16-Rb pathways). Specific factors in SASP (e.g., CXCL10, TRAIL) activate immune surveillance and recruit natural killer cells and cytotoxic T lymphocytes to clear tumor cells (Prasanna et al., 2021). On the other hand, senescence escape, observed in around 5% of p53-mutant tumors, enables epigenetic reprogramming (e.g., hypermethylation of the p16 promoter) and re-entry into the cell cycle, often conferring aggressive, therapy-resistant phenotypes (O’Sullivan et al., 2024). Therefore, while senescence serves as a defense against tumorigenesis, cancer cells can exploit this process to promote survival.

In summary, IR-induced cellular senescence is primarily driven by DNA damage and its downstream signaling. Understanding these mechanisms not only elucidates the effects of radiation on aging and cancer, but also informs strategies to mitigate radiation risks and harness senescence for therapeutic benefit.