ABSTRACT
Chromosomal aneuploidy has been associated with aging. However, whether and how chromosomal instability (CIN), a condition frequently seen in cancer cells in which chromosome missegregation occurs at a high rate, is associated with aging is not fully understood. Here, we found that primary fibroblasts isolated from aged mice (24 months old) exhibit an increased level of chromosome missegregation and micronucleation compared with that from young mice (2 months old), concomitant with an increased rate of aneuploid cells, suggesting the emergence of CIN. Reactive oxygen species were increased in fibroblasts from aged mice, which was accompanied with mitochondrial functional decline, indicating that they are under oxidative stress. Intriguingly, antioxidant treatments reduced chromosome missegregation and micronucleation rates in cells from aged mice, suggesting a link between oxidative stress and CIN. As a cause of CIN, we found that cells from aged mice are under replication stress, which was ameliorated by antioxidant treatments. Microtubule stabilization is a potential cause of CIN promoted by replication stress. Our data demonstrate the emergence of CIN with age, and suggest an unprecedented link between oxidative stress and CIN in aging.
INTRODUCTION
Genomic instability is one of the hallmarks of aging, which is characterized by the accumulation of genetic damage throughout life, such as point mutations, translocations, chromosomal gains and losses, telomere shortening and gene disruption (Lopez-Otin et al., 2013). Genomic instability is also a hallmark of cancer (Hanahan and Weinberg, 2011), and chromosomal gains and losses, known as aneuploidy, are found in the majority of cancer cells (Weaver and Cleveland, 2006). Aneuploidy in cancer cells is usually accompanied by chromosomal instability (CIN), a condition in which chromosome missegregation occurs at a high rate (Bakhoum and Compton, 2012; Gordon et al., 2012; Tanaka and Hirota, 2016). It has been reported that aneuploidy is also related to aging. For example, the Y chromosome is frequently lost in males with increasing age (Jacobs et al., 1963; Pierre and Hoagland, 1972). A longitudinal study comparing chromosomal contents in fibroblasts from the same individuals at 4–9-year intervals showed that aneuploidy increased with age (Mukherjee and Thomas, 1997). Increasing chromosome abnormalities including aneuploidy have also been shown in mice with advancing age (Lushnikova et al., 2011). However, whether CIN underlies the age-related aneuploidy has not been fully understood.
CIN can be directly evaluated by examining the rate of chromosome missegregation in either fixed samples or live-cell imaging, which is typically manifested as lagging chromosomes (single chromatids pulled towards both spindle poles via microtubules attaching to the single kinetochores) and chromosome bridges (connected chromosomes pulled towards both spindle poles via microtubules attaching to the respective kinetochores) in anaphase cells (Iemura et al., 2021; Kuniyasu et al., 2018). Observation of chromosome missegregation, though, has not been widely applied to evaluate CIN in non-cancerous tissues, partly due to scarcity of mitotic cells as well as difficulty to observe mitotic progression in vivo. Micronucleus formation is another index of CIN that is commonly used, and it has been demonstrated that the rate of micronucleation increases with age (Laffon et al., 2021). However, micronuclei are formed not solely by chromosome missegregation, but also can also be triggered by other causes, such as DNA damage (Fenech et al., 2011).
CIN arises through various deficits in mitotic processes, including the formation of erroneous kinetochore–microtubule attachments, defects in the spindle assembly checkpoint, centrosome amplification and cohesion defects (Tanaka and Hirota, 2016). It is known that hyperstabilization of microtubules attaching to kinetochores frequently underlies CIN in cancer cells, which hampers the efficient correction of erroneous kinetochore–microtubule attachments (Bakhoum et al., 2009a,b). CIN is caused not only by mitotic defects, but also by replication stress, which is the slowing or stalling of replication fork progression; this promotes DNA break formation and the appearance of acentric or fused chromosomes (Burrell et al., 2013). An association between CIN and aging has been emerging (Naylor and van Deursen, 2016; Rao et al., 2017). One example is mosaic variegated aneuploidy (MVA) syndrome, which is characterized by increased chromosome missegregation and a broad spectrum of clinical features including progeroid pathologies (Garcia-Castillo et al., 2008). BUBR1 (also known as BUB1B), a component of the spindle assembly checkpoint, is one of the genes disrupted in MVA, and mice containing hypomorphic alleles of Bub1b show CIN and progeroid phenotypes (Baker et al., 2004), whereas BUBR1 overexpression counteracts age-related aneuploidization and leads to an increased lifespan with attenuated aging-related phenotypes (Baker et al., 2013).
To address the presence and underlying cause of CIN in aging, we explored fibroblasts isolated from young (2 months old) and aged (24 months old) mice. Fibroblasts are widely used cells for primary culture to investigate various research topics including aging (Plikus et al., 2021; Salzer et al., 2018). We demonstrated that fibroblasts isolated from aged mice exhibit CIN. Inspired by the fact that mouse primary fibroblasts stop proliferation after a few passages under atmospheric oxygen (Parrinello et al., 2003), we found that oxidative stress induces CIN in mouse fibroblasts. We further found that fibroblasts isolated from aged mice are under oxidative stress even under physiological oxygen concentration due to mitochondrial functional decline. Oxidative stress in aged fibroblasts causes replication stress, which is expected to be a cause of CIN. Our data reinforce the presence of CIN in aged cells, and disclose the previously unrecognized link between oxidative stress and CIN in aging.
RESULTS
Fibroblasts isolated from aged mice exhibit CIN
To explore the alteration in chromosomal stability during aging, we focused on primary fibroblasts isolated from C57BL/6J mice at different ages. Fibroblasts migrated from minced mouse ears were grown under 3% O2 (Fig. S1A), which is a physiological oxygen concentration in vivo (Vaupel et al., 1989). Under atmospheric (20%) O2, cells stopped growing in a short period, as previously reported (data not shown; Parrinello et al., 2003; Seluanov et al., 2010). We found that growth of fibroblasts isolated from aged (24-month-old) mice was slower than that of fibroblasts isolated from young (2-month-old) mice (Fig. 1A). Cell cycle profiles determined by fluorescence-activated cell sorting (FACS) were comparable between fibroblasts from young and aged mice (Fig. 1B). As we found that tetraploid cell population, which is indicative of cellular transformation (Lanni and Jacks, 1998; Minn et al., 1996), increases through passages (Fig. S1B), we used cells at early passages (2–3 passages) and excluded cells with large nuclei, indicative of tetraploid cells for further analyses.
We first quantified the percentage of chromosome missegregation in fixed samples. Fibroblasts isolated from aged mice showed higher proportions of cells with lagging chromosomes and chromosome bridges compared with that from young mice (Fig. 1C). The percentage of cells containing micronuclei, which appear as a result of chromosome missegregation, was also higher in cells from aged mice compared with that from young mice (Fig. 1D). The percentage of binucleated cells was also higher in cells from aged mice, although the difference was not statistically significant (Fig. S1C). These data suggest that skin fibroblasts isolated from aged mice exhibit CIN, although the proportions of cells with chromosome missegregation and micronucleation were lower than those observed in cancer cell lines showing CIN (Iemura et al., 2021). We repeatedly quantified the micronucleation rates in independent experiments and confirmed its increase in fibroblasts from aged mice (Fig. S1D). Fibroblasts isolated from 12-month-old mice showed intermediate micronucleation rates (Fig. S1D). We further verified the findings in primary lung fibroblasts. Lung fibroblasts isolated from aged mice showed reduced growth compared with those from young mice (Fig. S1E). The proportion of cells with micronucleation increased in an age-dependent manner (Fig. S1F), consistent with the results in skin fibroblasts.
To address the consequence of CIN in cells from aged mice, we examined metaphase spreads from skin fibroblasts for the presence of aneuploidy. When we quantified the number of chromosomes, there was a significant increase in the proportion of metaphase cells with abnormal numbers of chromosomes in cells from aged mice (Fig. 1E), consistent with a previous report (Lushnikova et al., 2011). Both metaphases with fewer and more than 40 chromosomes increased, whereas the average number of chromosomes did not significantly change (Fig. S1G,H), showing that despite the marked increase in aneuploid cells in aged mice, the modal number was not altered. These data suggest that CIN in fibroblasts from aged mice leads to the appearance of aneuploid cells.
As it is known that senescent cells increase with age (McHugh and Gil, 2018), we examined the percentage of senescent cells by staining for senescence-associated (SA)-β-gal in skin fibroblasts. As expected, the proportion of SA-β-gal-positive cells increased with age, and increased further with passages (Fig. S2A). We observed the relationship between micronucleation and cellular senescence using SPiDER-βGal, a fluorescence senescence marker (Doura et al., 2016). Although not all the micronucleated cells were positive for SPiDER-βGal signal and vice versa, the presence of micronuclei and SPiDER-βGal signal were significantly correlated (Fig. S2B), implying that there is a relationship between micronucleation and cellular senescence. We also examined the proportion of myofibroblasts, which are differentiated from fibroblasts to facilitate tissue repair, and reported to increase in lung fibroblasts from aged mice (Paxson et al., 2011; Plikus et al., 2021). As shown in Fig. S2C, cells positive for α-smooth muscle actin (SMA), a marker for myofibroblasts, were increased with age, reaching nearly 30% in aged mice. When we observed the relationship between micronucleation and myofibroblasts, there was no correlation in cells from young and adult mice (Fig. S2D). In cells from aged mice, micronucleated cells were rather excluded from α-SMA-positive cells (Fig. S2D), suggesting that micronucleation is not related to differentiation to myofibroblasts.
To exclude the possibility that CIN observed in fibroblasts from aged mice is attributable to ex vivo culture, we examined the proportion of cells with micronuclei in tissue sections of mouse ear skin. The proportion of cells containing micronuclei was increased in tissue sections from aged mice compared with those from young mice (Fig. 1F), suggesting that CIN observed in skin fibroblasts from aged mice is not solely due to ex vivo culture. Collectively, these data indicate that mouse fibroblasts from aged mice exhibit a mild level of CIN.
Chromosome missegregation increases under elevated oxygen concentration
Mouse primary fibroblasts stop proliferation under 20% O2 after a couple of passages (Parrinello et al., 2003; Seluanov et al., 2010). As a cause of the reduced proliferation, we wondered whether mitotic defects increase under 20% O2. We performed live-cell imaging of mouse fibroblasts under 3% or 20% O2, which were cultured under 3% O2 before the imaging. We found that chromosome missegregation increased in cells from aged mice compared with that from young mice under 3% O2 (Fig. 2A–C), confirming the results of fixed cells. Intriguingly, cells with chromosome missegregation, both lagging chromosomes and chromosome bridges, increased significantly under 20% O2 in cell from young and aged mice (Fig. 2B,C). Mitotic duration was longer in cells from aged mice compared with that from young mice, and in cells observed under 20% O2 compared with that observed under 3% O2 (Fig. 2A,B,D), reflecting the increase in mitotic defects. Under 20% O2, cells that underwent chromosome missegregation spent longer time in mitosis, compared with cells that underwent proper segregation (Fig. 2E), showing that there was a correlation between mitotic defects and delayed mitotic progression. These data suggest that ambient oxygen perturbs proper chromosome segregation and mitotic progression in mouse primary fibroblasts.
Fibroblasts from aged mice are under oxidative stress
The possibility that exogenous oxidative stress increases chromosome missegregation led us to a further hypothesis that endogenous oxidative stress might be involved in CIN in cells from aged mice. To verify the possibility, we examined the presence of oxidative stress in fibroblasts from aged mice by detecting the level of reactive oxygen species (ROS). We found that the ROS level was higher under 20% O2 compared with the level under 3% O2 both in cells from young and aged mice, as expected (Fig. 3A). Intriguingly, the ROS level was higher in cells from aged mice compared with that from young mice, both under 3% and 20% O2 (Fig. 3A), suggesting that mouse primary fibroblasts isolated from aged mice are under oxidative stress. As ROS is mainly produced in mitochondria initially as superoxide (Murphy, 2009), we observed the level of superoxide in mitochondria using MitoSOX Red, and found that it was higher in cells from aged mice under 3% O2 (Fig. 3B). We further evaluated mitochondrial function by analyzing mitochondrial membrane potential, as its decline leads to respiratory-chain defects and enhanced ROS production (Passos et al., 2010; Vigneron and Vousden, 2010). As shown in Fig. 3C, mitochondrial membrane potential was reduced in cells from aged mice. These data suggest that mitochondrial functional decline results in increased ROS production in cells from aged mice.
Oxidative stress induces CIN in fibroblasts from aged mice
To examine the relationship between the increased ROS level and CIN in cells from aged mice, we observed the proportion of cells with micronuclei in the presence or absence of N-acetylcysteine (NAC), an antioxidant (Aruoma et al., 1989; Burgunder et al., 1989). Treatment with NAC under 3% O2 reduced the level of ROS in cells from aged mice, but not in cells from young mice (Fig. S3A). Consistent with the notion that mitochondrial functional decline causes increased ROS production, MitoSOX Red signal showed that NAC treatment reduces superoxide in mitochondria in cells from aged mice (Fig. S3B). As shown in Fig. 4A, the percentage of cells with micronuclei was reduced in the presence of NAC under 20% O2 both in young and aged cells, although it was not statistically significant in the former, verifying that increased oxidative stress is involved in the increase in the proportion of cells with micronuclei. Under 3% O2, the percentage of cells with micronuclei was reduced in cells from aged mice, but not in cells from young mice, implicating that oxidative stress is involved in the emergence of CIN in cells from aged mice (Fig. 4B). To examine the relationship between oxidative stress and CIN in detail, we performed live-cell imaging of mouse fibroblasts under 3% O2 in the presence or absence of NAC. We found that cells with chromosome missegregation, both lagging chromosomes and chromosome bridges, were reduced in cells from aged mice in the presence of NAC, but not in cells from young mice (Fig. 4C,D). Mitotic duration was also reduced when cells were treated with NAC, especially for aged mice (Fig. 4E), reflecting the improvement of mitotic progression. We also examined the effect of mitoquinone mesylate (MitoQ), a mitochondria-targeted antioxidant (Kelso et al., 2001). MitoQ reduced the level of ROS in cells from aged mice, as expected (Fig. S3C). MitoSOX Red signal was also reduced in cells from aged mice treated with MitoQ, although this was not statistically significant (Fig. S3D). We found that MitoQ reduced the percentage of cells with micronuclei in cells from aged mice (Fig. 4F), further confirming that oxidative stress induces CIN in cells from aged mice. Cells treated with MitoQ together with NAC did not show a further reduction in the proportion of cells with micronuclei compared to cells treated with NAC alone, whereas the reduction was small but significant compared to cells treated with MitoQ alone (Fig. S4A), implying that ROS generated not only in mitochondria but also at other sites are responsible for the induction of CIN in fibroblasts from aged mice.
Oxidative stress causes replication stress that promotes CIN in fibroblasts from aged mice
We investigated how oxidative stress causes CIN in fibroblasts from aged mice. CIN is induced not only by defects in the mitotic process but also by DNA damage in interphase caused by replication stress (Burrell et al., 2013; DePinho and Polyak, 2004). Therefore, we observed the presence of DNA damage in fibroblasts at different ages. The number of γ-H2AX foci, which represents the presence of DNA double strand breaks (DSBs) (Mah et al., 2010), increased in cells from aged mice (Fig. S4B), consistent with the previous reports showing that the frequency of γ-H2AX foci-containing cells increases in various mouse tissues with age (Wang et al., 2009) and oxidative stress increases γ-H2AX (Venkatachalam et al., 2017). Sensitivity to bleomycin, which causes DSBs, camptothecin (CPT), a DNA topoisomerase I inhibitor that produces single strand breaks that turn into DSBs when encountered by DNA replication machinery, and mitomycin C, a crosslinking agent that can cause DSBs in the process of crosslink repair (Lee et al., 2006), did not differ between cells from young and aged mice (Fig. S4C–E), suggesting that DSB repair per se is not defective in cells from aged mice. Interestingly, we found that the number of cells positive for 53BP1 foci, which represent DSBs under processing (Mirman and de Lange, 2020), was increased in cells from aged mice (Fig. 5A). These 53BP1 foci were large in size and few in number (see Fig. S4F), suggesting that they are 53BP1 nuclear bodies formed around DNA lesions generated by replication stress (Lukas et al., 2011). It has been reported that 53BP1 nuclear bodies are formed in G1 phase as a result of replication stress in the previous cell cycle, and often appear in a symmetrical manner between daughter cells (Lukas et al., 2011). When we observed the 53BP1 signal between daughter cells in G1 phase by inhibiting cytokinesis with cytochalasin B, there was a tendency that the fraction of daughter cells showing symmetrical 53BP1 nuclear bodies increased in cells from aged mice (Fig. 5B). An initial constituent of 53BP1 nuclear bodies involved in DNA damage response, activated ATM, as determined by auto-phosphorylation at S1981, also colocalized with the 53BP1 signal (Fig. 5B) (Lukas et al., 2011). Metaphase chromosome spreads showed an increase in chromosome breaks, indicative of structural chromosome aberrations arising through pre-mitotic defects including replication stress, in cells from aged mice (Fig. 5C). To clarify whether micronuclei in fibroblasts from aged mice were derived from whole chromosomes or acentric chromosome fragments caused by chromosome breaks, we observed micronuclei for the presence of CENP-A, a centromere marker. As shown in Fig. 5D, both CENP-A-positive and negative micronuclei increased in cells from aged mice, with more than half of the micronuclei being CENP-A-negative, implying that micronuclei in fibroblasts from aged mice contain both whole chromosomes and acentric chromosome fragments, which are expected to be caused by replication stress. We also observed the presence of ultrafine bridges (UFBs), which are thin DNA threads connecting sister chromatids in anaphase that can result from replication stress (Baumann et al., 2007; Chan et al., 2007, 2009). The percentage of anaphase cells with UFBs, which were detected by an antibody against PICH (also known as ERCC6L), was significantly increased in cells from aged mice (Fig. 5E), corroborating that cells from aged mice are under replication stress. To address the relationship between replication stress and CIN, we treated cells with a low dose of aphidicolin, which inhibits DNA synthesis and causes replication stress. As shown in Fig. 5F,G, aphidicolin treatment increased the percentage of cells with 53BP1 nuclear bodies and UFBs in cells from both young and aged mice, confirming that replication stress induces the formation of 53BP1 nuclear bodies. Importantly, aphidicolin treatment significantly increased the percentage of cells containing micronuclei in cells from young and aged mice (Fig. 5H), supporting the notion that replication stress causes CIN. To validate the causal relationship between replication stress and CIN in cells from aged mice, we supplemented cells with nucleosides, which partially rescues replication stress (Burrell et al., 2013). In cells from aged mice, the percentage of cells with 53BP1 nuclear bodies and UFBs were reduced (Fig. 5I,J), confirming that replication stress was rescued. Intriguingly, the percentage of cells containing micronuclei was reduced in cells from aged mice supplemented with nucleosides (Fig. 5K). Mitotic indices did not change in the presence of nucleosides both in cells from young and aged mice (Fig. S4G), excluding the possibility that decreased mitoses is responsible for these reductions. These data suggest that replication stress is a cause of CIN in cells from aged mice.
Microtubule stabilization is related to CIN induced by replication stress caused by oxidative stress in fibroblasts from aged mice
To reveal the relationship between oxidative stress and replication stress, we examined whether the formation of 53BP1 nuclear bodies is affected by NAC treatment. As shown in Fig. 6A, NAC treatment reduced the proportion of 53BP1 nuclear body-positive cells under 20% O2 both in cells from young and aged mice, although it was not statistically significant in the former. Under 3% O2, the percentage of cells with 53BP1 nuclear bodies was reduced in cells from aged mice, but not in cells from young mice (Fig. 6B), suggesting that oxidative stress is involved in the accrual of replication stress in cells from aged mice. MitoQ also reduced the percentage of cells with 53BP1 nuclear bodies in cells from aged mice (Fig. 6C). Cells treated with MitoQ together with NAC showed a small but significant reduction in the proportion of 53BP1-positive cells compared to cells treated with MitoQ alone (Fig. S4A), implying that ROS generated not only in mitochondria, but also at other sites, are responsible for the induction of replication stress in fibroblasts from aged mice. Moreover, NAC treatment reduced the percentage of anaphase cells with UFBs in cells from aged mice (Fig. 6D), confirming that oxidative stress causes replication stress in cells from aged mice.
It was recently reported that replication stress causes CIN through stabilization of microtubules, which reduces the correction efficiency of erroneous kinetochore–microtubule attachments (Wilhelm et al., 2019). To examine the stability of microtubules, we quantified the intensity of spindle microtubules that are resistant to a 10-min cold treatment on ice, which depolymerizes unstable microtubules (Iemura and Tanaka, 2015). Intensity of spindle microtubules was comparable between cells from young and aged mice at room temperature (Fig. S4H). However, the spindle microtubule intensity after 10 min-cold treatment was significantly higher in cells from aged mice (Fig. 6E), suggesting the tendency that microtubules are stabilized in cells from aged mice. If microtubule stabilization caused CIN in cells from aged mice, an increase in microtubule dynamics would be expected to alleviate CIN. To increase microtubule dynamics, we treated cells with UMK57, a potentiator of MCAK (also known as KIF2C), a motor protein with microtubule-depolymerizing activity (Orr et al., 2016). As shown in Fig. 6F, UMK57 treatment reduced the percentage of cells containing micronuclei in cells from aged mice, corroborating that microtubule stabilization is related to CIN. The percentage of cells containing 53BP1 nuclear bodies did not differ in the presence or absence of UMK57 (Fig. 6G), showing that replication stress is not downstream of CIN. Cells treated with UMK57 in combination with NAC showed no further reduction in the proportion of 53BP1-positive cells and micronucleation compared to cells treated with NAC alone (Fig. S4A), demonstrating that increasing microtubule dynamics through UMK57 treatment does not cause an additive effect with NAC treatment on replication stress and micronucleation. In contrast, the proportion of cells containing micronuclei was further reduced in cells treated with NAC and UMK57 compared to cells treated with UMK57 alone (Fig. S4A), suggesting that microtubule stabilization partially contributes to micronucleation downstream of oxidative stress in cells from aged mice. Collectively, these data suggest that oxidative stress causes replication stress, which might be related to CIN in cells from aged mice through microtubule stabilization.
DISCUSSION
Our data demonstrate that a mild level of CIN is found in skin fibroblasts from aged mice, which is related to oxidative stress accompanied with mitochondrial functional decline. To our knowledge, this is the first report that has clarified the link between aging, oxidative stress and CIN. We also found that oxidative stress causes replication stress, which might promote CIN. Our data corroborate the presence of CIN in cells from aged individuals that underlies age-related aneuploidy.
We observed mitotic progression of mouse primary fibroblasts by live-cell imaging under 3% and 20% O2, which enabled us to directly assess CIN and specify oxidative stress as its cause. It was known that compared to human fibroblasts, which do not spontaneously immortalize in culture, mouse embryonic fibroblasts stop dividing after only 10–15 doublings before immortalization (Wright and Shay, 2000). It was later found that this is due to a high sensitivity of mouse fibroblasts to atmospheric oxygen, which causes DNA damage (Parrinello et al., 2003). Our data extended the finding and suggest that the slow proliferation of mouse fibroblasts under atmospheric oxygen is partly due to mitotic defects caused by oxidative stress. An increase in the proportion of aneuploid cells under atmospheric oxygen has also been reported in mouse hematopoietic cells (Liu et al., 2012). Although human cells are more resistant to oxidative stress than mouse cells, mitotic defects have also been observed when they were challenged with hydrogen peroxide (D'Angiolella et al., 2007; Wang et al., 2017). It has also been reported that oxidative stress causes chromosome missegregation in Drosophila oocytes (Perkins et al., 2016) and human–hamster hybrid cells (Limoli and Giedzinski, 2003). Our finding that elevated oxidative stress with age is related to mitochondrial dysfunction is corroborated by previous findings (Capel et al., 2005; Cui et al., 2012; Forster et al., 1996; Gilmer et al., 2010; Kudryavtseva et al., 2016; Navarro et al., 2002). Enhanced mitochondrial ROS production accompanied by the decline of mitochondrial membrane potential is also consistent with previous reports (Ma et al., 2009; Passos et al., 2010). By showing that chromosome missegregation in cells from aged mice under 3% O2 was reduced by an antioxidant treatment, we clearly demonstrated that oxidative stress promotes CIN in cells from aged mice. Supporting our data, CIN induction by mitochondrial oxidative stress in mouse embryonic fibroblasts as well as reduced aneuploidization upon an antioxidant treatment in mouse hematopoietic cells from aged mice has been previously reported (Liu et al., 2012; Samper et al., 2003). Building on these previous studies, our study clearly demonstrates the link between aging, oxidative stress and CIN in the same model system.
We found that cells with 53BP1 foci are increased in cells from aged mice. These foci are supposedly so-called 53BP1 nuclear bodies, which are formed in G1 phase in response to replication stress in the previous cell cycle (Lukas et al., 2011). Together with the increase in UFBs in cells from aged mice, as well as the reduction of the 53BP1 foci and UFBs by antioxidant treatments, our data suggest that oxidative stress causes replication stress in cells from aged mice. It is known that replication stress is a cause of CIN (Wilhelm et al., 2020). We confirmed that aphidicolin treatment, which causes replication stress, increased the rate of micronucleation in cells from both young and aged mice. Furthermore, the micronucleation rate was reduced in cells from aged mice supplemented with nucleosides, implying that replication stress might be a cause of CIN in cells from aged mice. Regarding how oxidative stress causes mitotic defects, several mechanisms have been proposed, such as overriding of the SAC and inhibition of Aurora A-involved spindle formation (D'Angiolella et al., 2007; Wang et al., 2017). We found that spindle microtubules were hyperstabilized in cells from aged mice, and the micronucleation rate was reduced by increasing microtubule dynamics. As microtubule hyperstabilization is a cause of CIN commonly seen in cancer cells (Bakhoum et al., 2009a,b), and a cause of CIN induced by mild replication stress (Wilhelm et al., 2019), it can account for the emergence of CIN under replication stress in cells from aged mice. Increased number of stable kinetochore–microtubule attachments in skin fibroblasts from octogenerians were also observed in a previous study (Barroso-Vilares et al., 2020). The same group reported that increased chromosome missegregation in skin fibroblasts from octogenerians correlates with repression of FoxM1, a transcription factor involved in mitotic gene expression (Macedo et al., 2018). How repression of FoxM1 is related to oxidative stress for the induction of CIN warrants further study. As the rates of chromosome missegregation and micronucleation of cells from aged mice did not reduce to the level of cells from young mice upon antioxidant treatments, factors other than oxidative stress might also be involved in the aging-related CIN. Another important issue is to verify whether the mitotic defects found in in vitro culture faithfully recapitulates in vivo situation, as it was shown that tissue architecture supports chromosome segregation fidelity (Knouse et al., 2018).
The consequences of CIN in aging are not fully understood. It has been known that oxidative stress and resulting DNA damage induce cellular senescence (d'Adda di Fagagna, 2008). It was also proposed that mitotic defects in cells from octogenerians results in aneuploidization, which triggers a full senescence phenotype (Macedo et al., 2018). In line with the finding, we found a correlation between the presence of micronuclei and the SPiDER-βGal signal. As fibroblasts are heterogenous populations including myofibroblasts (Plikus et al., 2021), detailed analysis of the fates of cells that underwent chromosome missegregation is necessary. Besides senescence, CIN is a hallmark of cancer, and oxidative stress is known to be a cause of CIN in cancer (Kudryavtseva et al., 2016). Therefore, the link we found between oxidative stress and CIN in cells from aged mice might be related to age-dependent oncogenesis. Interestingly, oncogene-induced CIN in mouse embryonic fibroblasts was attenuated by antioxidants, implicating oxidative stress in induction of CIN for neoplastic transformation (Woo and Poon, 2004). Although it is now recognized that a certain level of ROS plays a role in cellular homeostasis (Lopez-Otin et al., 2013), keeping the ROS level within a correct range might be a way to maintain chromosomal stability and suppress oncogenesis.
MATERIALS AND METHODS
Mice
For aged C57BL/6J male mice, mice obtained from Charles River Laboratories were bred in groups of one or two under the specific pathogen-free conditions (ad libitum access to food and water, 12 h light, 12 h dark cycle, light on at 08:00). For young C57BL/6JJcr male mice, 8-week-old mice were obtained from CLEA Japan Inc. at the time of each experiment. All experimental procedures conformed to the Regulations for Animal Experiments and Related Activities at Tohoku University, and were reviewed by the Institutional Laboratory Animal Care and Use Committee of Tohoku University, and finally approved by the President (2020AcA-011).
Cell culture and synchronization
Ears or lungs of C57BL/6J male mice [2 months (6–9 weeks) old, 12 months (48–54 weeks) old, and 24 months (94–102 weeks) old] were used to isolate fibroblasts. Briefly, tissues were minced and small fragments were incubated twice with DMEM/Ham's F-12 with L-glutamine, sodium pyruvate, and HEPES (Nacalai, 08460-95) containing 0.13 Wünsch units/ml LiberaseTM Research Grade (Merck, 5401119001) for 1 h at 37°C in 5% CO2, and cultured in DMEM/Ham's F-12 with L-glutamine, sodium pyruvate and HEPES supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2 and 3% O2. To enrich mitotic cells, mouse primary fibroblasts were treated with 2 mM of thymidine (Wako) for 24 h, followed by treatment with 10 µM of RO-3306 (Tokyo Chemical Industry, R0201) for 12 h, then released for indicated periods before fixation.
Flow cytometry
Cells were washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4), pelleted (450 g for 5 min), and fixed with 70% ethanol for 2 h at 4°C. After being washed twice with cold PBS, cells were incubated with 250 µg/ml RNase A (Merck) for 1 h at 37°C. The cells were stained with 50 µg/ml propidium iodide for 30 min at 4°C. The samples were immediately analyzed by flow cytometry with a Cytomics FC500 machine (Beckman Coulter). Doublets and debris were excluded by appropriate gating using the SSC-A and FSC-A. The cell cycle phase distribution was determined using MultiCycle AV (Phoenix Flow Systems) software.
Reagents and antibodies
Bleomycin (Fujifilm Wako Chemicals, 028-07801) was used at 2.5, 5 and 10 μg/ml. Camptothecin (Merck, C9911) was used at 25, 50 and 100 nM. Mitomycin C (Nacalai, 20898-21) was used at 200, 400 and 600 ng/ml. Cytochalasin B (Fujifilm Wako Chemicals, 030-17551) was used at 0.72 µg/ml. N-acetyl-L-cysteine (NAC; Merck, A7250) and mitoquinone mesylate (MitoQ; Selleck, M36008) were used at 5 mM and 500 nM, respectively. MG-132 (Merck, M8699) was used at 20 µM. UMK57 (AOBIOS, AOB8668) was used at 100 nM. Aphidicolin (Fujifilm Wako Chemicals, 011-09811) was used at 200 nM. For nucleoside supplementation, a mix of deoxycytidine (Tokyo Chemical Industry, D3583), deoxyadenosine (Tokyo Chemical Industry, D0046), thymidine (Tokyo Chemical Industry, T0233) and deoxyguanosine (Tokyo Chemical Industry, D0052) was applied at 20 µM for 48 h. A rabbit polyclonal antibody for 53BP1 (NOVUS Biologicals, NB100-304) and PICH (ERCC6L; Proteintech, 15688-1-AP), and rabbit monoclonal antibody for CENP-A (Cell Signaling Technology, 2047S) were used for immunofluorescence at 1:1000. A mouse monoclonal antibody for α-tubulin (B5-1-2; Merck, T5168), α-smooth muscle actin (α-SMA, 1A4; Abcam, ab7817), phospho ATM (Ser1981; Rockland, 200-301-400), and phospho-Histone H2A.X (Ser139 (γ-H2AX), JBW301; Merck, 05-636) were used for immunofluorescence at 1:1000.
Metaphase chromosome spreads
Cells were treated with 2 mM of thymidine for 24 h, followed by treatment with 10 µM of RO-3306 for 12 h, then treated with 10 µM of MG-132 and 2 µM of nocodazole (Merck, M1404) for 2 h. Mitotic cells collected by a shake-off were swollen hypotonically by adding 4 volumes of tap water for 5 min, and then fixed with Carnoy's solution (3:1 methanol:acetic acid). The cell suspension was spotted onto slide glasses, dried and mounted with VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, H-1200). Images were captured on an Olympus IX-83 inverted microscope (Olympus) controlled by cellSens Dimension 1.18 (Olympus) using a ×100 1.40 numerical aperture (NA) UPlanS Apochromat oil objective lens (Olympus).
Immunofluorescence analysis
Immunofluorescence analysis was performed according to a previous report (Iemura and Tanaka, 2015). Briefly, cells were grown on a glass coverslip and fixed with 3% paraformaldehyde in PBS for 15 min at room temperature, and permeabilized with 1% Triton X-100 in PBS for 5 min. Fixed cells were incubated with primary antibodies for more than 12 h at 4 °C, washed three times with PBS supplemented with 0.02% Triton X-100, and incubated with secondary antibodies coupled with Alexa-Fluor-488 or -594 (Thermo Fisher Scientific, 1:2000) for 1 h at room temperature. Antibody incubations were performed in PBS supplemented with 0.02% Triton X-100. After final washes, cells were mounted with VECTASHIELD Mounting Medium with DAPI. Z-image stacks were captured in 0.2 or 0.27 µm increments on an Olympus IX-83 inverted microscope controlled by cellSens Dimension 1.18 using a ×100 1.40 or ×60 1.35 NA UPlanS Apochromat oil objective lens. Deconvolution was performed when necessary. Image stacks were projected and saved as TIFF files. All cells analyzed were selected from non-overlapping fields.
HE staining
Mouse skin tissues were embedded using OCT compound (Sakura Finetek Japan), and cut into 14 µm sections using Leica CM3050 S Cryostat. Slice sections were dried on MAS-coated glass slides (Matsunami Glass Ind.) and fixed with 10% formalin in methanol for 1 min. Fixed sections were stained with Hematoxylin solution (Mayer's, Modified, Abcam) for 5 min, washed with tap water, and then stained with 0.16% Eosin Y Solution (Muto Pure Chemicals Co.) in 60% ethanol, pH 5.0 for 5 min, and washed with tap water. Stained sections were dehydrated in an ethanol series and with xylene, and mounted with EUKITT mounting medium (Kindler). Images were captured on a BZ-9000 (Keyence) using CFI Plan Apo ×40 objective lens (Nikon), and saved as TIFF files.
SA-β-gal staining
Cells were grown in six-well plates (Violamo), and SA-β-gal activity was detected using a cellular senescence kit (OZ Biosciences, GXS0003), according to the manufacturer's instructions. Briefly, the cells were washed twice with PBS, fixed in the fixation buffer at room temperature for 15 min, washed three times with PBS, and stained using the β-galactosidase staining solution for 12 h at 37°C. Photo images were taken using a Nikon Coolpix P340 through an Olympus IX-83 using a 20×0.45 NA LUCPlanFLN objective lens (Olympus). To observe the presence of micronuclei together with SA-β-gal activity, cells were treated with 1 µM SPiDER-βGal (Dojindo, SG02) for 15 min, and fixed with 3% paraformaldehyde in PBS for 15 min at room temperature. Then cells were stained with DAPI for 20 min. Images were captured on an Olympus IX-83 inverted microscope controlled by cellSens Dimension 1.18 using a 60×1.35 NA Plan Apochromat oil objective lens.
MTT assay
Cells were grown in 96-well plates (Violamo) at 5000 cells/well for 12 h, and treated with drugs at different concentrations for 48 h. Then cells were incubated with 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT, Nacalai) for 4 h, followed by incubation with 5% SDS in 5 mM HCl for 12 h. The absorbance values at the wavelength of 550 nm and 690 nm were measured using SpectraMax® M2e (Molecular Devices).
ROS detection
Cells were grown in 3.5 cm glass bottom dishes (MatTek), and ROS was measured using CellROX Oxidative Stress Reagents (Molecular Probes, C10444), according to the manufacturer's instructions. Briefly, the cells were stained with 5 µM CellROX green reagents with 2 µM verapamil hydrochloride (Fujifilm Wako Chemicals, 222-00781) for 30 min at 37°C in 5% CO2 and 3% O2 before imaging. Superoxide in mitochondria was detected using MitoSOX Red (Molecular Probes, M36008), according to the manufacturer's instructions. Briefly, the cells were stained with 5 µM MitoSOX Red with 2 µM verapamil hydrochloride for 10 min at 37°C in 5% CO2 and 3% O2 before imaging. Images were captured on an Olympus IX-83 inverted microscope controlled by cellSens Dimension 1.18 using ×60 1.40 NA Plan Apochromat oil objective lens. Image stacks were projected and saved as TIFF files.
Mitochondrial membrane potential assay
Mitochondrial membrane potential was measured using JC-10 (AAT Bioquest, 22204) according to the manufacturer's instructions. Briefly, the cells were grown in 3.5 cm glass bottom dishes, and stained with 1× JC-10 with 2 µM verapamil hydrochloride for 30 min at 37°C in 3% O2 before imaging. Images were captured on an Olympus IX-83 inverted microscope controlled by cellSens Dimension 1.18 using a 60×1.40 NA Plan Apochromat oil objective lens. Image stacks were projected and saved as TIFF files.
Live-cell imaging
Cells were grown in 12- or 6-well plates (Violamo), and treated with 5 nM SiR-DNA (Cytoskeleton, CY-SC007) and 2 µM verapamil hydrochloride for 4 h at 37°C in 5% CO2 and 3% O2 before imaging. Recordings were made every 2.5 min for 48 h using the Celldiscoverer 7 live-cell imaging system (Carl Zeiss) controlled by ZEN 2.6 software (Carl Zeiss) at 37°C in 5% CO2 and either 3% or 20% O2 using a 20×0.7 NA Plan Apochromat objective lens (Carl Zeiss). Images were analyzed using ZEN 2.6.
Statistical analysis
The Mann–Whitney U-test was used for comparison of dispersion, and a two-sided unpaired t-test was used for comparisons of average. A two-sided F-test validated the dispersibility of each category before the Student's t-test. If the result of the F-test was an unequal variance, a significant difference between samples was validated by a two-sided Welch's t-test. A one-way ANOVA test was used with the Tukey–Kramer posthoc test for comparisons between all groups showing normal distribution. The Kolmogorov–Smirnov test verified the normality of data distribution for each group before the one-way ANOVA test. If the result of the Kolmogorov–Smirnov test was non-nominal distribution, the significant differences between all groups were validated by the Kruskal–Wallis test, which was used with Steel–Dwass posthoc test. For comparisons between the single group and multi groups showing normal distribution, Dunnett's posthoc test was used after a one-way ANOVA test. A χ-squared test was used for comparison between the measured value and theoretical value. All statistical analyses were performed with EZR (Kanda, 2013), which is a graphical user interface for R [R Core Team, R: A language and environment for statistical computing, https://www.R-project.org/, (2018)]. More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics. Samples for analysis in each data set were acquired in the same experiment, and all samples were calculated at the same time for each data set.
Acknowledgements
The authors thank members of the K.T. laboratory for discussions, and A. Harata and H. Sato for technical assistance. The authors also thank the Laboratory for Animal Resources and the Center of Research Instruments in the Institute of Development, Aging and Cancer, Tohoku University for technical support.
Footnotes
Author contributions
Conceptualization: K.T.; Methodology: G.C., K.I.; Validation: K.I., K.T.; Formal analysis: G.C., Z.L.; Investigation: G.C., Z.L.; Resources: K.T.; Data curation: G.C., K.I.; Writing - original draft: K.T.; Writing - review & editing: G.C., K.I., K.T.; Visualization: G.C., K.I.; Supervision: K.T.; Project administration: K.T.; Funding acquisition: G.C., K.I., K.T.
Funding
This work was supported by Japan Society for the Promotion of Science (JSPS; KAKENHI grant numbers 26640067, 15H04368, 16K14604, 18H02434, 22K19283), Ministry of Education, Culture, Sports, Science and Technology (MEXT; KAKENHI grant numbers, 26114702, 18H04896), and grants from the Takeda Science Foundation to K.T.; JSPS KAKENHI grant numbers 16H06635, 18K15234, 20K16295; Uehara Memorial Foundation, Kanae Foundation for the Promotion of Medical Science, Yamaguchi Ikuei Foundation, and Gonryo Medical Foundation to K.I. and Japan Science and Technology Agency (JST)-supported university fellowships towards the creation of science technology innovation (grant number JPMJFS2102) to G.C. G.C. was supported by an Advanced Graduate School Doctoral Fellowship and Global Hagi Scholarship for doctoral students from Tohoku University, and a Otsuka Toshimi Scholarship.
Data availability
All relevant data can be found within the article and its supplementary information.
Peer review history
The peer review history is available online at https://journals.biologists.com/jcs/lookup/doi/10.1242/jcs.260688.reviewer-comments.pdf.
References
Competing interests
The authors declare no competing or financial interests.