Cigarette smoke contains thousands of chemicals and many chemicals impose health hazards including carcinogenic, cardiovascular and respiratory effects (Li and Hecht, 2022; Talhout et al., 2011). Cigarette smoke is also associated with the induction of cellular senescence, a state with irreversible cell cycle arrest (Nyunoya et al., 2006). Senescent cells have altered metabolism, morphology, and secretory profile called the senescence-associated secretory phenotype (SASP), which includes pro-inflammatory cytokines, chemokines, and proteases (Coppé et al., 2008). The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, an important pathway for the induction for SASP (Takahashi et al., 2018), is activated by exposure to cigarette smoke (Khan et al., 2024; Nascimento et al., 2019; Ueda et al., 2023), providing another evidence for the induction of cellular senescence by cigarette smoke.

DNA damages are known to induce premature cellular senescence (Yousefzadeh et al., 2021) and accumulating evidence suggests cigarette smoke induces DNA damages (Alexandrov et al., 2016; Khan et al., 2024; Ueda et al., 2023; Yang et al., 1999). Our previous study demonstrated the repair inhibitory effect of cigarette smoke (Yang and Ibuki, 2018). With the combination of DNA damage inducing capability and repair inhibition, it is tempting to imagine DNA damage would accumulate after repeated exposure to cigarette smoke. Persistent DNA damage triggers signaling cascades that drives cells into apoptosis or senescence to avoid replicating a damaged genome and thus promote aging (Yousefzadeh et al., 2021). Moreover, DNA fragments derived from nucleus and mitochondria DNA damage are accumulated in cytosol, leading to activation of cGAS-STING pathway (Ueda et al., 2023).

The genomic DNA of eukaryotes is highly organized and packed into chromatin. The nucleosome, the basic unit of chromatin, is formed by 146 bp of DNA wrapping around an octameric core of histone proteins (Hauer and Gasser, 2017). The state of histone proteins, critical for chromatin compaction, is far from static, but rather dynamically regulated. Histones are post-translationally modified and evicted at sites of DNA damage to facilitate the recruitment of DNA damage repair factors: reduction in cellular histone levels as much as 20–40 % occurred in response to DNA damage by proteolytic degradation (Hauer et al., 2017). The change of histone levels is also reported in senescent cells (Feser et al., 2010; Lopez et al., 2012; Lowe et al., 2020; O'Sullivan et al., 2010). Histone levels were reduced in senescent cells induced by various means, owing to suppressed histone biosynthesis and accompanied by the loss of proper regulation of gene expression (Lowe et al., 2020). Conversely, elevating histone expression in aging yeast cells extended their life span (Feser et al., 2010). However, alteration of histone level during cigarette smoke-induced senescence has not been elucidated.

Among histone proteins, the histone H2AX protein is well-known for its function in the DNA damage response (DDR). Upon DNA damage, the serine residue in the C-terminus (Ser139) is phosphorylated by PI3K-like kinases including ataxia-telangiectasia mutated (ATM), ATM and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) (Burma et al., 2001; Stiff et al., 2004; Ward and Chen, 2001) to form γ-H2AX, which then acts as a platform for the binding of DNA repair factors, thus playing an critical role for efficient DNA repair and genome integrity (Celeste et al., 2002; Paull et al., 2000; Podhorecka et al., 2010). H2AX does not seem to be essential for survival, but mice lacking H2AX showed phenotypes associated with chromosomal instability, repair defects, and impaired recruitment of several repair factors to the damaged sites (Celeste et al., 2002). On the other hand, γ-H2AX is also commonly used as markers of senescence since the initiation of cellular senescence requires irreparable DNA lesions and sustained DDR (Hernandez-Segura et al., 2018; Kobashigawa et al., 2019; Siddiqui et al., 2015).

How would H2AX affect the induction of cigarette smoke-induced cellular senescence? Absence of H2AX could result in two opposite consequences. The depletion of H2AX would lead to the depletion of DDR, which may prevent the induction of cellular senescence. Conversely, decreased H2AX level may prevent efficient repair of cigarette smoke-induced DNA damages; as a result, DNA damages may persist even longer, which may promote cellular senescence. To elucidate the role of H2AX during cigarette smoke-induced cellular senescence, we used cigarette sidestream smoke (CSS) extract and normal diploid fibroblast from human skin ASF-4-1, which has a limited life span and is frequently used as a model system for cellular aging (Kaji et al., 2009). Our results indicate that DSBs induced by CSS exposure could be the main cause for CSS-induced cellular senescence and that H2AX plays a protective role against DSBs-induced cellular senescence.