Dysregulation of the low-level replication stress response in transformed cell lines

Dysregulation of the low-level replication stress response in transformed cell lines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dysregulation of the low-level replication stress response in transformed cell lines Sandrine Ragu, Elodie Dardillac, Sylvain Caillat, Jean-Luc Ravanat, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6155647/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract The canonical DNA damage response (cDDR) maintains genome stability, involving DNA synthesis arrest. However, unchallenged cells proliferate when they are continually exposed to low-level/endogenous replication stress. We previously characterized a noncanonical response specific to nonblocking replication stress, i.e. low-level stress (LoL-DDR), in primary cells. Although LoL-DDR generates replication stress-induced ROS (RIR), it prevents the accumulation of premutagenic 8-oxo-guanine (8-oxoG). Primary cells control RIR production via NADPH oxidases. Increasing the severity of replication stress above a threshold triggers the cDDR, leading to cell cycle arrest and RIR suppression, resulting in a peak-shaped dose response for RIR production. Here, we show that the LoL-DDR is dysregulated in cancer cell lines, which exhibit the following differences compared with primary cells: 1- RIR are not detoxified under high-level stress, resulting in a continuous increase in the dose‒response curve of RIR production; 2- RIR are not produced by NADPH oxidases; 3- replication stress favors the accumulation of the premutagenic 8-oxoG. Moreover, using an in vitro breast cancer progression model, we show that LoL-DDR dysregulation occurs at an early stage of cancer progression. Since, conversely, ROS trigger replication stress this establishes a “vicious circle” replication-stress/ROS that continuously jeopardizes genome integrity that should fuel and amplify tumorigenesis. Biological sciences/Molecular biology Biological sciences/Molecular biology/Dna damage and repair Biological sciences/Molecular biology/Dna damage and repair/Dna damage response Genome instability Replication stress DDR Mutagenesis ROS Cancer initiation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Genome is daily assaulted by various exogenous or endogenous attacks that jeopardize its integrity, leading to cell death, inflammation, aging or tumorigenesis. To cope with these stresses, cells have developed the DNA damage response (DDR) that coordinates a network of pathways allowing the faithful transmission of the genome. In response to genotoxic stress, the well-documented “canonical” DDR (cDDR) triggers cell cycle checkpoints that slow or arrest cell cycle progression 1 . Deficiency in this cDDR drives genetic instability, which is frequently associated with cancer susceptibility 1 – 7 . Notably, the cDDR has been shown to be activated in response to replication stress at the pre/early steps of senescence and tumorigenesis 2 – 4 , 8 , 9 , suggesting that this type of stress might participate in oncogenesis. In primary cells, a threshold of stress intensity must be reached to induce the cDDR 10 . In the absence of sufficiently strong stress to fully induce the cDDR, which leads to the arrest of DNA replication and cell cycle progression, cells are still routinely exposed to inevitable low stresses/endogenous stresses that can potentially also threaten genome stability. Such low/endogenous stress can include replicative and oxidative stresses. Indeed, replication fork progression can be hindered by endogenous obstacles (transcription, endogenous damage, particular DNA structures, proteins tightly bound to DNA, etc.) 11 – 14 . Additionally, reactive oxygen species (ROS), which are spontaneous byproducts of cell metabolism, can generate premutagenic bases and can alter replication dynamics 15 – 17 . Despite chronic exposure to low-level/endogenous stresses, cells proliferate and replicate their genome. This suggests that such low stresses escape detection by the cDDR. Recently, we discovered and characterized a “noncanonical” DDR that specifically occurs in response to low-level stress in primary human cells, and we defined this response as the low-level stress DDR (LoL-DDR); this response does not lead to the arrest of DNA replication but still protects genome integrity 10 . We have shown that primary human cells adapt their response to the severity of replicative stress in two distinct phases: full activation of the cDDR requires that stress reach an intensity threshold; below this threshold, cell proliferation is not blocked, but cells produce ROS, namely, replication-induced ROS (RIR), which then activate the detoxification program, reducing the level of the premutagenic base 8-oxo-guanine (8-oxoG) in the genome 10 . Because ROS can negatively affect genome integrity, RIR production is precisely controlled by cells, and RIR production follows known trends in primary human cells. 1- The dose response of RIR follows a peak-shaped curve; RIR production increases with replication stress intensity until a threshold is reached, and above this threshold (when the stress intensity increases), the cDDR is induced, cell progression is arrested, and RIR are scavenged, resulting in a peak-shaped dose‒response curve for RIR. 2- RIR production is controlled by the NADPH oxidases DUOX1 and DUOX2. 3- RIR prevents the accumulation of 8-oxoG in the genome in an adaptive manner 10 . Therefore, in primary cells, RIR appear to function as secondary messengers of an autonomous response to preserve genome stability, specifically in response to low-level/endogenous stress (LoL-DDR), which do not lead to the arrest of replication progression. These findings reveal the precise regulation of primary cell responses to stress severity. This response is critical because, in contrast to severe stress conditions, cells are exposed to chronic pernicious and inevitable low-level/endogenous stress conditions each day throughout their lifespan. Because genetic instability is a hallmark of cancer cells 7 , 18 , 19 , we addressed the question of whether the LoL-DDR might be dysregulated in transformed cell lines. Here, we show that in immortalized/transformed cells, 1- RIR production follows a dose‒response curve, continuously increasing with stress severity, even at higher doses, which differs from with the peak-shaped dose‒response curve observed in primary fibroblasts in which RIR production decreases in response to higher stress; 2- RIR are not produced by NADPH oxidases in transformed cells, unlike in primary cells; and 3-replication stress in transformed/immortalized cells leads to the accumulation of the premutagenic lesion 8-oxoG into the genome, which differs from findings in primary fibroblasts. Moreover, in an in vitro cellular model, the LoL-DDR appears to be altered at an early stage of breast cancer progression. These data reveal the dysregulation of the LoL-DDR in transformed cells, which might reveal the following potential sequence of events: replication stress induces ROS production, which results in the accumulation of premutagenic lesions in the genome; reciprocally, ROS induce replication stress 15 , 17 . Therefore, these data reveal a “vicious circle”. This cycle may maintain and amplify genetic instability. This vicious circle” might be critical for the initiation/progression of cancer, which is consistent with the fact that transformed cells have highly active replication programs and high levels of ROS and with previous description of replication stress in vivo in pre/early stages of oncogenesis 2 – 4 , 8 , 9 . RESULTS The dose‒response curve of RIR production is altered in transformed cell lines. First, we measured the intracellular levels of ROS by FACS using the 2’,7’-dichlorofluorescein diacetate (DCFDA) fluorescent probe ( Fig. 1 A), which allows convenient analysis of the dose response to a treatment that induces replication stress 10 . In primary fibroblasts or epithelial cells, the RIR dose‒response curve exhibited a peak shape; maximum RIR production was observed in response to 250 µM hydroxyurea (HU), which is an inhibitor of ribonucleotide reductase that results in an imbalance of the nucleotide pools and in fine replication stress. At higher replication stress (HU > 250 µM) a cell cycle progression and the level of RIR decreases (see 10 and the scheme in Fig. 1 B). Importantly, this type of peak-shaped curve has been described in 4 different primary fibroblast strains and in primary epithelial cells, for each of the individual repeat of the experiment, at the same doses of replication stress-inducing drugs, attesting the robustness of the response in primary human cells 10 . In parallel with the experiments with human primary fibroblasts described above 10 , we treated different transformed cell lines with increasing doses of drugs that generate replication stress. After exposure to HU, all three human cell lines, namely, the U2OS (human osteosarcoma), RKO (human colon cancer with wild-type p53), and RG37 (SV40-tranormed human fibroblasts 20 ) cell lines, responded similarly; that is, these cells lines exhibited a gradual increase in RIR production, but in contrast to primary cells 10 , there was no decrease in RIR product after treatment with higher doses of HU ( Fig. 1 C and compared with Fig. 1 B and 10 ) . A hamster cell line, namely, the V79 cell line (a p53-mutated lung Chinese hamster cancer cell line), also exhibited the same behavior (Fig. 1 D). This finding shows that the same dose-dependent continuous increase in RIR production is observed in several types of cancer cell lines, even those with different p53 statuses and those from different species (human and hamster). These curves are different from those observed in primary cells (compare Fig. 1 B to Fig. 1 C–D and 10 ). In primary fibroblasts, RIR are abolished at high HU doses (500 or 1000 µM) when DNA replication is blocked 10 . In transformed cells, RIR production still increased at such high doses (Fig. 1 C-D), i.e., even when replication was strongly abrogated and when the cells accumulated in the S or G2/M phase (Fig. 1 E). RIR production thus appears to be dysregulated in transformed cells compared with primary cells, reflecting a change in regulation of the Lol-DDR. Therefore, the dose‒response curves of RIR production represent a reliable diagnosis marker of changes in the LoL-DDR. Using this readout, we then analyzed other parameters. We next investigated another replication stress inducer, namely, aphidicholin (APH), which is an inhibitor of replicative polymerase. Exposure of primary cells to APH also resulted in a peak-shaped response in RIR production, which peaked after treatment with APH doses between 0.6 and 1.2 µM and then decreased after treatment with higher doses (Fig. 1 B and 10 ). In contrast to primary cells, but similar to HU treatment, exposure of all 3 human transformed cell lines to APH led to gradual continuous RIR production, with increased ROS production observed even after treatment with higher doses (Fig. 2 A). Together, these data suggest that RIR product actually occurs due to replication stress but that transformed cell lines respond differently than primary cells. Telomerase-immortalized retinal pigment epithelium (RPE) cells exhibited a dose‒response curve similar to that of the cell lines described above, i.e., a continuous and progressive increase in RIR production was observed, although the level of stimulation was lower (Fig. 2 B). However, human fibroblasts that were immortalized with telomerase (BJ-hTert) exhibited a response that was intermediated relative to the responses of primary and transformed cells (Fig. 2 C). Indeed, after treatment with higher HU doses, the RIR levels neither increased nor decreased; rather, RIR levels plateaued (Fig. 2 C). This finding shows that while the LoL-DDR is actually altered in immortalized cells, such alteration differs in cells of different tissue types. Nevertheless, these data suggest that changes in the LoL-DRR might occur in the early stage of cell transformation. Dysregulation of the LoL-DDR occurs at an early stage of cancer progression in an in vitro cell model. To address the above question, we used a cell culture model of breast cancer progression 21 , 22 . This model was established with primary epithelial cells (HUMECs), telomerase-immortalized HUMECs (HME-hTert cells), HME-hTert cells expressing the SV40 large T antigen (HMLE-SV40), and HMLE-SV40 cells expressing the V-RAS oncogene. Notably, RIR levels peaked in primary breast epithelial cells after treatment with 250 µM HU (Fig. 2 D and 10 ), which is similar to what was observed in primary fibroblasts (see Fig. 1 B and 10 ). The dose‒response curves of the immortalized HME-hTert cells lost their peak shape, and RIR production progressively increased with increasing dose (Fig. 2 D), similar to what was observed in the other transformed cell lines (compare with Fig. 1 C-D). Notably, the change in dose response occurred as soon as the HMR-hTert cells, and the curve increased with increasing dose. This response is similar to that of RPE-hTert cells (compare with Fig. 2 B), another epithelial cell line. These data suggest the potential dysregulation of the LoL-DDR in an early stage of breast cancer progression. RIR are not produced by NADPH oxidases in immortalized/transformed cell lines. In primary fibroblasts, RIR are secondary messengers of the LoL-DDR, and they protect genome stability in an adaptive way 10 . As such, RIR production is precisely controlled by the cellular enzymes NADPH oxidases 10 . The exposure of primary human cells (fibroblasts as well as epithelial mammary cells) to diphenylene iodonium chloride (DPI), which inhibits all NADPH oxidases 23 , suppressed RIR production 10 . In contrast to its effect on primary cells, DPI did not reduce RIR levels in transformed cells, and it even show a trend to induce ROS production in cells that were not exposed to HU (Fig. 3 A). Moreover, in the breast cancer progression model used here, although RIR production was indeed abrogated by DPI in primary epithelial cells (HUMEC), RIR production became resistant to DPI as early as the HME-hTert step (Fig. 3 B), i.e., in the same stage as when the alteration in the dose‒response curves of RIR production was observed. Collectively, these data confirm the dysregulation of the LoL-DDR in an early stage of breast cancer progression, at least in in vitro cellular models. The total cellular levels of RIR were not affected by DPI, which suggests that no or only a marginal portion of RIR might be produced by NADPH oxidases and that ROS from other cellular sources account for a large portion of the cellular RIR levels in transformed cell lines. Replication stress leads to the accumulation of oxidative DNA lesions in transformed cell lines. In primary cells, RIR prevent the accumulation of oxidative DNA damage in the nuclear genome 10 . Given the alteration in the LoL-DDR that was observed in transformed cells, we assessed whether RIR might differentially impact the accumulation of oxidative damage in the nuclear genome. To address this question, levels of the main premutagenic oxidized base lesion, namely, 8-oxoG, which is a marker of genotoxic oxidative stress, were quantified through isotope dilution high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC–MS/MS) 24 . In primary cells, the frequency of 8-oxoG decreased at doses that induced peak RIR production (50 and 250 µM HU), and then, 8-oxoG did not further accumulate after treatment with higher HU doses (Fig. 4 A) 10 . In contrast, in transformed cells, the frequency of 8-oxoG in genomic DNA gradually increased upon HU treatment in a dose-dependent manner (Fig. 4 B), which was consistent with the dose‒response curves of RIR production (compare Fig. 4 B with Fig. 1 C-D). Consistently, in TERT-immortalized fibroblasts (BJ-Tert), the level of 8-oxoG increased and plateaued after treatment with the HU dose that caused the RIR levels to plateau (compare Fig. 4 C with Fig. 2 C). Collectively, these data support the concept that the LoL-DDR, which prevents the accumulation of premutagenic lesions in primary cells in response to low-level/endogenous replication stress 10 , is altered in transformed cells. DISCUSSION Cells are routinely exposed to stresses that compromise the integrity of their genome. The cDDR allows cells to cope with these genotoxic stresses. Schematically, the cDDR stops cell cycle progression, providing time and cofactors access to the repair machineries. This enables the resumption of the cell cycle with restored DNA matrices 25 – 27 . Defects in the cDDR lead to genome instability and cancer predisposition 2 , 4 – 7 , 25 . However, in the absence of strong exogenous stresses, cells can divide and replicate their genome despite the fact that they are faced with endogenous stress conditions. Recently, we showed that a threshold of stress intensity is required to induce the cDDR in primary human cells 10 . We identified and characterized a noncanonical DDR specific to low-level stress (LoL-DDR) in primary human cells, which results in RIR generation but prevents the accumulation of the premutagenic lesion 8-oxoG in the genome in an adaptive manner 10 . This pathway is characterized by the fact that: 1-the level of RIR decreases at high level stress intensity, 2- RIR are produced by the NADPH-oxidases, and 3- the prevention against the accumulation of pre-mutagenic oxidative lesions such as 8-oxoG 10 . Here, we show that the protective LoL-DDR is dysregulated in transformed cell lines. 1-RIR still accumulate under high replication stress conditions, which differs from RIR levels in primary cells; 2- cellular RIR production occurs independently of NADPH oxidase control; and 3- the accumulation of premutagenic 8-oxoG in the cell genome. In addition, experiments using an in vitro cellular model of breast cancer progression suggested that dysregulation of the LoL-DDR occurs in an early stage of cancer progression. An increase in premutagenic lesions with increased replication stress should induce mutagenesis, resulting in genetic instability that could foster cancer initiation and progression as well as the appearance of neoantigens. Importantly, the process described here has been shown to occur in cell lines with wild-type p53 (RKO, U2OS, and RPE-tert cells) as well as in cells with mutated p53 (V79 cells) or in which p53 is trapped by the SV40 large T antigen (RG37 cells). Thus RIR production in immortalized/transformed cell lines occurs independently of p53. These data are consistent with the fact that LoL-DDR dysregulation occurs in an early stage of cancer progression, as shown here. As summarized in Fig. 5 , in primary noncancer cells, nonblocking, low-level replication stress induces the LoL-DDR, which protects genome stability; increasing the stress intensity turns off the LoL-DDR but induces the cDDR, which maintains genetic stability but in a different way. Notably, all the factors that play pivotal roles in controlling the LoL-DDR, are lost in different types of tumors 28 – 34 . In immortalized cells, the LoL-DDR is dysregulated in the early stage of cancer progression, and replication stress induces ROS production even at high levels of stress, leading to the accumulation of premutagenic lesions in the genome. Since, ROS cause replication stress 15 , 17 , this triggers a vicious cycle, which maintains and amplifies genetic instability and thus should play an important role in cancer etiology in the both initiation and progression stages (Fig. 5 ). METHODS Cell culture and treatments The cells were grown at 37°C with 5% CO 2 in DMEM supplemented with 10% fetal calf serum (FCS). Primary human mammary epithelial cells (HuMECs) were obtained from Thermo Fisher Scientific and cultured according to the manufacturer’s recommendations. HuMEC derivatives (HME, HMLE, and HMLER cells) were obtained from Professors Alain Puisieux (Lyon, France) and Robert Weinberg (Boston, USA) and were cultured in 1:1 DMEM/HAM-F12 medium (Gibco, Life Technologies) supplemented with 10% FBS (Lonza Group, Ltd.), 100 U/ml penicillin‒streptomycin (Invitrogen), 10 ng/ml human epidermal growth factor (EGF) (Sigma), 0.5 µg/ml hydrocortisone (Sigma) and 10 µg/ml insulin (Sigma). Primary fibroblasts were exposed to HU, APH or CPT for 72 h at 37°C. For antioxidant treatment, primary fibroblasts were exposed to 2 mM NAC (Sigma‒Aldrich, St. Louis, MO, USA) for 72 h. Measurement of cellular ROS production by FACS analysis. Cellular ROS production was measured with a CM-H2DCFDA (2’,7’-dichlorofluorescein diacetate) (Life Technologies, USA) or dihydrorhodamine 123 (DHR 123) (Sigma) assay kit according to the manufacturer’s protocol. Approximately 10 5 cells/well were plated into 6-well plates and incubated at 37°C in 5% CO 2 . After 2 days, the cells were washed with PBS and incubated with 10 µM CM-H2DCFDA or DHR 123 in DMEM supplemented with 1% FBS for 45 min at 37°C in the dark. The cells were trypsinized and resuspended in DMEM supplemented with 1% FBS. The pelleted cells were washed again, and the live cells were resuspended in PBS and analyzed on a BD Accuri C6 flow cytometer (BD Biosciences, San Diego, CA) equipped with an FL1 laser (515–545 nm). The data are presented as the mean percentages of four independent experiments. Cell cycle analysis and BrdU incorporation. Cells were incubated in culture medium supplemented with or without HU for 72 h at 37°C, and 5-bromo-2-deoxyuridine (BrdU, Sigma) was added to the culture medium at a final concentration of 10 µM and incubated for 30 min. Pelleted cells were detached with trypsin, fixed with 80% ethanol, and resuspended in 30 mM HCl/0.5 mg/ml pepsin. BrdU was immunofluorescently labeled with a mouse anti-BrdU antibody (DAKO, clone Bu20a) and a fluorescein-conjugated donkey anti-mouse antibody (Life Technologies), and the cells were stained with propidium iodide (PI; 25 µg/ml) in the presence of ribonuclease A (50 µg/ml). Flow cytometry analyses were performed using an Accuri C6 flow cytometer (BD Biosciences). 8-OxoG measurement. Genomic DNA was extracted and enzymatically digested using an optimized protocol that minimizes DNA oxidation during the procedure 35 . Then, 8-oxoG levels were quantified using isotope dilution high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-MS/MS) as previously described 24 ; 15 N 5 -8-oxoG was used as the internal standard. In addition to the mass spectrometric detector, the system is equipped with a UV detector that is set to 260 nm to measure the quantity of normal nucleosides. The results are expressed as the number of 8-oxoG lesions per million normal nucleosides. Statistical analyses. Statistical analyses were performed with GraphPad Prism 10. P < 0.05 was considered to indicate a statistically significant difference. Declarations ACKNOWLEDGMENTS. We thank Corinne Dupuy (Gustave Roussy Cancer Institute) for helpful discussions and advice on NADPH oxidases. Author Contributions. Conceptualization: BSL; funding acquisition: BSL; investigation: SR, ED, SC; methodology: SR, BSL, SC, and J-LR; 8-oxoG dosage experiments: SC and J-L R; project administration: BSL; resources: BSL; supervision: BSL; validation: SR, ED, J-LR, and BSL; writing the original draft: BSL; and writing – reviewing & editing: all the authors. Data availability. All data generated or analysed during this study are included in this published article. If needed, more details or explanations can be available from the corresponding author on reasonable request. Declaration of Interests. The authors declare that they have no conflicts of interest. Funding This work was supported by grants from the Institut National du Cancer (PLBIO21-072, PLBIO24-194), La Ligue Nationale Contre Le Cancer (ARN thérapeutiques), Ligue régionale contre le cancer (comité Ile de France), (ITMO Cancer (PCSI 2022), and Fondation ARC (ARCPJA2022060005157). References Jackson, S. P. & Bartek, J. 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Cite Share Download PDF Status: Published Journal Publication published 06 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 11 Apr, 2025 Reviews received at journal 10 Apr, 2025 Reviews received at journal 31 Mar, 2025 Reviewers agreed at journal 23 Mar, 2025 Reviewers agreed at journal 21 Mar, 2025 Reviewers invited by journal 19 Mar, 2025 Editor assigned by journal 19 Mar, 2025 Editor invited by journal 12 Mar, 2025 Submission checks completed at journal 12 Mar, 2025 First submitted to journal 04 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6155647","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":427861186,"identity":"1ac8e824-6dda-4851-bcdb-5ac811915b28","order_by":0,"name":"Sandrine Ragu","email":"","orcid":"","institution":"Université de Paris, INSERM U1016, UMR 8104 CNRS, Institut Cochin","correspondingAuthor":false,"prefix":"","firstName":"Sandrine","middleName":"","lastName":"Ragu","suffix":""},{"id":427861187,"identity":"bb4a379b-5de8-4230-9c6b-03b356885dbf","order_by":1,"name":"Elodie Dardillac","email":"","orcid":"","institution":"Université de Paris, INSERM U1016, UMR 8104 CNRS, Institut Cochin","correspondingAuthor":false,"prefix":"","firstName":"Elodie","middleName":"","lastName":"Dardillac","suffix":""},{"id":427861188,"identity":"6fa32dd4-09af-431d-af41-1153815ef014","order_by":2,"name":"Sylvain Caillat","email":"","orcid":"","institution":"Univ. Grenoble Alpes, CEA, CNRS, Grenoble INP, IRIG","correspondingAuthor":false,"prefix":"","firstName":"Sylvain","middleName":"","lastName":"Caillat","suffix":""},{"id":427861189,"identity":"4cd20502-0ab5-4f97-aa60-9e347b08dce7","order_by":3,"name":"Jean-Luc Ravanat","email":"","orcid":"","institution":"Univ. Grenoble Alpes, CEA, CNRS, Grenoble INP, IRIG","correspondingAuthor":false,"prefix":"","firstName":"Jean-Luc","middleName":"","lastName":"Ravanat","suffix":""},{"id":427861190,"identity":"5cb6d802-4596-4b44-a420-490dff836909","order_by":4,"name":"Bernard S. Lopez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYBACAwjFzMAHpiskoOI2RGhhA9NnYFrSiNXC2AYTx6PFnL332YOfe6wZ2Nh7Dz78Oc8iT7699+kGhoR7OLVY9hw3N+x5ls7AxnMu2UBym0SxwZnjZjcYEopxO+xGGpsEz4HDDGwSOWYShtskEjdIpLHdYPyRgFvL/Wdskn8gWsx/JM6RSJw//xkb0BY8Wm6wsUnDbGE42CCR2AAUwavFsieNTVrmQDoPyC+SDceADjsDdFgCHi3m7MfYJN8csJbjB4bYxx81dYnz24+x3fiARwsM8IARHBDWANM1CkbBKBgFowAbAADo403JCdmhGQAAAABJRU5ErkJggg==","orcid":"","institution":"Université de Paris, INSERM U1016, UMR 8104 CNRS, Institut Cochin","correspondingAuthor":true,"prefix":"","firstName":"Bernard","middleName":"S.","lastName":"Lopez","suffix":""}],"badges":[],"createdAt":"2025-03-04 15:38:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6155647/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6155647/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-05172-0","type":"published","date":"2025-06-06T15:57:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78513879,"identity":"ad2f325e-47cd-42fe-8bd7-b2530042fced","added_by":"auto","created_at":"2025-03-14 10:23:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":333281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrimary and transformed cells differentially produce ROS in response to replication stress. A\u003c/strong\u003e. HU- or APH-induced ROS production was monitored using the DCFDA fluorescent probe and FACS analysis. The shift in the fluorescence peak reveals the induction of intracellular ROS production.\u003cstrong\u003e B. \u003c/strong\u003eTypical peak-shaped dose-response curve of primary fibroblasts or epithelial cells following replication stress \u003csup\u003e10\u003c/sup\u003e.\u003cstrong\u003e C. \u003c/strong\u003eDose response of ROS induction in three different human cell lines after 48 h of exposure to HU. \u003cstrong\u003eD.\u003c/strong\u003e Dose response of ROS induction in V79 hamster cells after 48 h of exposure to HU.\u003cstrong\u003e E\u003c/strong\u003e. Impact of exposure to HU on the cell cycle distribution. The data from at least three independent experiments are presented as the mean (± SEM) level of ROS production normalized to that of the control. The non-parametric Kruskal-Wallis test was used.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6155647/v1/0eb5b31c00ca13c8c5612b47.png"},{"id":78513131,"identity":"ebe4bd57-8939-466b-85e5-3eba32c86996","added_by":"auto","created_at":"2025-03-14 10:15:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":105224,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRIR production under different conditions. A. \u003c/strong\u003eImpact of APH exposure on three different human transformed cell lines. \u003cstrong\u003eB. \u003c/strong\u003eRIR production in response to HU in RPE-hTert immortalized cells.\u003cstrong\u003e C.\u003c/strong\u003e RIR production in response to HU in human fibroblast-hTert immortalized cells. \u003cstrong\u003eD. \u003c/strong\u003eRIR production in response to HU in the \u003cem\u003ein vitro \u003c/em\u003ebreast cancer progression model. The data from at least three independent experiments are presented as the mean (± SEM) level of ROS production normalized to that of the control. The non-parametric Kruskal-Wallis test was used.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6155647/v1/35a87dc8d2d869954c08d245.png"},{"id":78513877,"identity":"67a6bb41-61eb-4a26-99bb-75a0b734e35d","added_by":"auto","created_at":"2025-03-14 10:23:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRIR are resistant to NADPH oxidase inhibition in immortalized/transformed cells. A. \u003c/strong\u003eU2OS and SV40-transformed human fibroblasts\u003cstrong\u003e. B. \u003c/strong\u003e\u003cem\u003eIn vitro \u003c/em\u003ebreast cancer progression model.\u003cstrong\u003e \u003c/strong\u003eThe data from at least three independent experiments are presented as the mean (± SEM) level of ROS production normalized to that of the control.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6155647/v1/48eb5e19be2061b824f53f3b.png"},{"id":78513135,"identity":"a21575bf-c04d-4a08-9239-245e94c0ca2f","added_by":"auto","created_at":"2025-03-14 10:15:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":48687,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e8-oxoG accumulation A. \u003c/strong\u003ePrimary fibroblasts, data from \u003csup\u003e10\u003c/sup\u003e.\u003cstrong\u003e B\u003c/strong\u003e. U2OS- and SV40-transformed human fibroblasts.\u003cstrong\u003e C\u003c/strong\u003e. human telomerase-immortalized fibroblasts BJ-Tert. The data from three independent experiments are presented as the mean (± SEM) level of ROS production normalized to that of the control. The non-parametric Kruskal-Wallis test was used.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6155647/v1/c50b4f8b57303546b578ff7a.png"},{"id":78513133,"identity":"b03df67e-d205-4a31-ada5-c34f86084724","added_by":"auto","created_at":"2025-03-14 10:15:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferent responses of cancer and primary cells to replication stress.\u003c/strong\u003e Upper panel: Below a particular threshold of stress intensity, primary cells induce the LoL-DDR, which is a noncanonical stress response that produces RIR, triggering detoxification in an adaptive way. Above this threshold, the cells induce the cDDR and detoxify RIR \u003csup\u003e10\u003c/sup\u003e. Both responses protect genome stability. Lower panel: In cancer cells, these responses are dysregulated, and ROS levels continuously increase with increasing stress intensity, resulting in the accumulation of premutagenic oxidative lesions (8-oxoG). Since ROS induce replication stress \u003csup\u003e15,17\u003c/sup\u003e, this reciprocally triggers vicious circle of replication stress and ROS production, which promote genome instability, potentially fueling tumor initiation and progression.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6155647/v1/c1fb35a58fcbbe4b84e30158.png"},{"id":84242638,"identity":"5b744d62-a542-4d69-a87c-3ad81567ea92","added_by":"auto","created_at":"2025-06-09 16:10:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1341479,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6155647/v1/c3ceb278-a20e-443e-a1e1-22cdb048b656.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dysregulation of the low-level replication stress response in transformed cell lines","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eGenome is daily assaulted by various exogenous or endogenous attacks that jeopardize its integrity, leading to cell death, inflammation, aging or tumorigenesis. To cope with these stresses, cells have developed the DNA damage response (DDR) that coordinates a network of pathways allowing the faithful transmission of the genome. In response to genotoxic stress, the well-documented \u0026ldquo;canonical\u0026rdquo; DDR (cDDR) triggers cell cycle checkpoints that slow or arrest cell cycle progression \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Deficiency in this cDDR drives genetic instability, which is frequently associated with cancer susceptibility \u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Notably, the cDDR has been shown to be activated in response to replication stress at the pre/early steps of senescence and tumorigenesis \u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, suggesting that this type of stress might participate in oncogenesis.\u003c/p\u003e \u003cp\u003eIn primary cells, a threshold of stress intensity must be reached to induce the cDDR \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In the absence of sufficiently strong stress to fully induce the cDDR, which leads to the arrest of DNA replication and cell cycle progression, cells are still routinely exposed to inevitable low stresses/endogenous stresses that can potentially also threaten genome stability. Such low/endogenous stress can include replicative and oxidative stresses. Indeed, replication fork progression can be hindered by endogenous obstacles (transcription, endogenous damage, particular DNA structures, proteins tightly bound to DNA, etc.) \u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Additionally, reactive oxygen species (ROS), which are spontaneous byproducts of cell metabolism, can generate premutagenic bases and can alter replication dynamics \u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Despite chronic exposure to low-level/endogenous stresses, cells proliferate and replicate their genome. This suggests that such low stresses escape detection by the cDDR. Recently, we discovered and characterized a \u0026ldquo;noncanonical\u0026rdquo; DDR that specifically occurs in response to low-level stress in primary human cells, and we defined this response as the low-level stress DDR (LoL-DDR); this response does not lead to the arrest of DNA replication but still protects genome integrity \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe have shown that primary human cells adapt their response to the severity of replicative stress in two distinct phases: full activation of the cDDR requires that stress reach an intensity threshold; below this threshold, cell proliferation is not blocked, but cells produce ROS, namely, replication-induced ROS (RIR), which then activate the detoxification program, reducing the level of the premutagenic base 8-oxo-guanine (8-oxoG) in the genome \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Because ROS can negatively affect genome integrity, RIR production is precisely controlled by cells, and RIR production follows known trends in primary human cells. 1- The dose response of RIR follows a peak-shaped curve; RIR production increases with replication stress intensity until a threshold is reached, and above this threshold (when the stress intensity increases), the cDDR is induced, cell progression is arrested, and RIR are scavenged, resulting in a peak-shaped dose‒response curve for RIR. 2- RIR production is controlled by the NADPH oxidases DUOX1 and DUOX2. 3- RIR prevents the accumulation of 8-oxoG in the genome in an adaptive manner \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Therefore, in primary cells, RIR appear to function as secondary messengers of an autonomous response to preserve genome stability, specifically in response to low-level/endogenous stress (LoL-DDR), which do not lead to the arrest of replication progression. These findings reveal the precise regulation of primary cell responses to stress severity. This response is critical because, in contrast to severe stress conditions, cells are exposed to chronic pernicious and inevitable low-level/endogenous stress conditions each day throughout their lifespan.\u003c/p\u003e \u003cp\u003eBecause genetic instability is a hallmark of cancer cells \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, we addressed the question of whether the LoL-DDR might be dysregulated in transformed cell lines. Here, we show that in immortalized/transformed cells, 1- RIR production follows a dose‒response curve, continuously increasing with stress severity, even at higher doses, which differs from with the peak-shaped dose‒response curve observed in primary fibroblasts in which RIR production decreases in response to higher stress; 2- RIR are not produced by NADPH oxidases in transformed cells, unlike in primary cells; and 3-replication stress in transformed/immortalized cells leads to the accumulation of the premutagenic lesion 8-oxoG into the genome, which differs from findings in primary fibroblasts. Moreover, in an \u003cem\u003ein vitro\u003c/em\u003e cellular model, the LoL-DDR appears to be altered at an early stage of breast cancer progression.\u003c/p\u003e \u003cp\u003eThese data reveal the dysregulation of the LoL-DDR in transformed cells, which might reveal the following potential sequence of events: replication stress induces ROS production, which results in the accumulation of premutagenic lesions in the genome; reciprocally, ROS induce replication stress \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Therefore, these data reveal a \u0026ldquo;vicious circle\u0026rdquo;. This cycle may maintain and amplify genetic instability. This vicious circle\u0026rdquo; might be critical for the initiation/progression of cancer, which is consistent with the fact that transformed cells have highly active replication programs and high levels of ROS and with previous description of replication stress \u003cem\u003ein vivo\u003c/em\u003e in pre/early stages of oncogenesis \u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eThe dose‒response curve of RIR production is altered in transformed cell lines.\u003c/b\u003e First, we measured the intracellular levels of ROS by FACS using the 2\u0026rsquo;,7\u0026rsquo;-dichlorofluorescein diacetate (DCFDA) fluorescent probe \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), which allows convenient analysis of the dose response to a treatment that induces replication stress \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In primary fibroblasts or epithelial cells, the RIR dose‒response curve exhibited a peak shape; maximum RIR production was observed in response to 250 \u0026micro;M hydroxyurea (HU), which is an inhibitor of ribonucleotide reductase that results in an imbalance of the nucleotide pools and \u003cem\u003ein fine\u003c/em\u003e replication stress. At higher replication stress (HU\u0026thinsp;\u0026gt;\u0026thinsp;250 \u0026micro;M) a cell cycle progression and the level of RIR decreases (see \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and the scheme in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Importantly, this type of peak-shaped curve has been described in 4 different primary fibroblast strains and in primary epithelial cells, for each of the individual repeat of the experiment, at the same doses of replication stress-inducing drugs, attesting the robustness of the response in primary human cells \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn parallel with the experiments with human primary fibroblasts described above \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, we treated different transformed cell lines with increasing doses of drugs that generate replication stress. After exposure to HU, all three human cell lines, namely, the U2OS (human osteosarcoma), RKO (human colon cancer with wild-type p53), and RG37 (SV40-tranormed human fibroblasts \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e) cell lines, responded similarly; that is, these cells lines exhibited a gradual increase in RIR production, but in contrast to primary cells \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, there was no decrease in RIR product after treatment with higher doses of HU \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC \u003cb\u003eand\u003c/b\u003e compared with Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e. A hamster cell line, namely, the V79 cell line (a p53-mutated lung Chinese hamster cancer cell line), also exhibited the same behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). This finding shows that the same dose-dependent continuous increase in RIR production is observed in several types of cancer cell lines, even those with different p53 statuses and those from different species (human and hamster). These curves are different from those observed in primary cells (compare Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB \u003cb\u003eto\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026ndash;D and \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e). In primary fibroblasts, RIR are abolished at high HU doses (500 or 1000 \u0026micro;M) when DNA replication is blocked \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In transformed cells, RIR production still increased at such high doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D), i.e., even when replication was strongly abrogated and when the cells accumulated in the S or G2/M phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). RIR production thus appears to be dysregulated in transformed cells compared with primary cells, reflecting a change in regulation of the Lol-DDR. Therefore, the dose‒response curves of RIR production represent a reliable diagnosis marker of changes in the LoL-DDR. Using this readout, we then analyzed other parameters.\u003c/p\u003e \u003cp\u003eWe next investigated another replication stress inducer, namely, aphidicholin (APH), which is an inhibitor of replicative polymerase. Exposure of primary cells to APH also resulted in a peak-shaped response in RIR production, which peaked after treatment with APH doses between 0.6 and 1.2 \u0026micro;M and then decreased after treatment with higher doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e). In contrast to primary cells, but similar to HU treatment, exposure of all 3 human transformed cell lines to APH led to gradual continuous RIR production, with increased ROS production observed even after treatment with higher doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Together, these data suggest that RIR product actually occurs due to replication stress but that transformed cell lines respond differently than primary cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTelomerase-immortalized retinal pigment epithelium (RPE) cells exhibited a dose‒response curve similar to that of the cell lines described above, i.e., a continuous and progressive increase in RIR production was observed, although the level of stimulation was lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, human fibroblasts that were immortalized with telomerase (BJ-hTert) exhibited a response that was intermediated relative to the responses of primary and transformed cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Indeed, after treatment with higher HU doses, the RIR levels neither increased nor decreased; rather, RIR levels plateaued (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This finding shows that while the LoL-DDR is actually altered in immortalized cells, such alteration differs in cells of different tissue types. Nevertheless, these data suggest that changes in the LoL-DRR might occur in the early stage of cell transformation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDysregulation of the LoL-DDR occurs at an early stage of cancer progression in an\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003ecell model.\u003c/b\u003e To address the above question, we used a cell culture model of breast cancer progression \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. This model was established with primary epithelial cells (HUMECs), telomerase-immortalized HUMECs (HME-hTert cells), HME-hTert cells expressing the SV40 large T antigen (HMLE-SV40), and HMLE-SV40 cells expressing the V-RAS oncogene. Notably, RIR levels peaked in primary breast epithelial cells after treatment with 250 \u0026micro;M HU (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e), which is similar to what was observed in primary fibroblasts (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e). The dose‒response curves of the immortalized HME-hTert cells lost their peak shape, and RIR production progressively increased with increasing dose (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), similar to what was observed in the other transformed cell lines (compare with Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). Notably, the change in dose response occurred as soon as the HMR-hTert cells, and the curve increased with increasing dose. This response is similar to that of RPE-hTert cells (compare with Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), another epithelial cell line. These data suggest the potential dysregulation of the LoL-DDR in an early stage of breast cancer progression.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRIR are not produced by NADPH oxidases in immortalized/transformed cell lines.\u003c/b\u003e In primary fibroblasts, RIR are secondary messengers of the LoL-DDR, and they protect genome stability in an adaptive way \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. As such, RIR production is precisely controlled by the cellular enzymes NADPH oxidases \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The exposure of primary human cells (fibroblasts as well as epithelial mammary cells) to diphenylene iodonium chloride (DPI), which inhibits all NADPH oxidases \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, suppressed RIR production \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In contrast to its effect on primary cells, DPI did not reduce RIR levels in transformed cells, and it even show a trend to induce ROS production in cells that were not exposed to HU (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Moreover, in the breast cancer progression model used here, although RIR production was indeed abrogated by DPI in primary epithelial cells (HUMEC), RIR production became resistant to DPI as early as the HME-hTert step (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), i.e., in the same stage as when the alteration in the dose‒response curves of RIR production was observed. Collectively, these data confirm the dysregulation of the LoL-DDR in an early stage of breast cancer progression, at least in \u003cem\u003ein vitro\u003c/em\u003e cellular models.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe total cellular levels of RIR were not affected by DPI, which suggests that no or only a marginal portion of RIR might be produced by NADPH oxidases and that ROS from other cellular sources account for a large portion of the cellular RIR levels in transformed cell lines.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReplication stress leads to the accumulation of oxidative DNA lesions in transformed cell lines.\u003c/b\u003e In primary cells, RIR prevent the accumulation of oxidative DNA damage in the nuclear genome \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Given the alteration in the LoL-DDR that was observed in transformed cells, we assessed whether RIR might differentially impact the accumulation of oxidative damage in the nuclear genome. To address this question, levels of the main premutagenic oxidized base lesion, namely, 8-oxoG, which is a marker of genotoxic oxidative stress, were quantified through isotope dilution high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC\u0026ndash;MS/MS) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In primary cells, the frequency of 8-oxoG decreased at doses that induced peak RIR production (50 and 250 \u0026micro;M HU), and then, 8-oxoG did not further accumulate after treatment with higher HU doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In contrast, in transformed cells, the frequency of 8-oxoG in genomic DNA gradually increased upon HU treatment in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), which was consistent with the dose‒response curves of RIR production (compare Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB with Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). Consistently, in TERT-immortalized fibroblasts (BJ-Tert), the level of 8-oxoG increased and plateaued after treatment with the HU dose that caused the RIR levels to plateau (compare Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC \u003cb\u003ewith\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollectively, these data support the concept that the LoL-DDR, which prevents the accumulation of premutagenic lesions in primary cells in response to low-level/endogenous replication stress \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, is altered in transformed cells.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCells are routinely exposed to stresses that compromise the integrity of their genome. The cDDR allows cells to cope with these genotoxic stresses. Schematically, the cDDR stops cell cycle progression, providing time and cofactors access to the repair machineries. This enables the resumption of the cell cycle with restored DNA matrices \u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Defects in the cDDR lead to genome instability and cancer predisposition \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, in the absence of strong exogenous stresses, cells can divide and replicate their genome despite the fact that they are faced with endogenous stress conditions. Recently, we showed that a threshold of stress intensity is required to induce the cDDR in primary human cells \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. We identified and characterized a noncanonical DDR specific to low-level stress (LoL-DDR) in primary human cells, which results in RIR generation but prevents the accumulation of the premutagenic lesion 8-oxoG in the genome in an adaptive manner \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This pathway is characterized by the fact that: 1-the level of RIR decreases at high level stress intensity, 2- RIR are produced by the NADPH-oxidases, and 3- the prevention against the accumulation of pre-mutagenic oxidative lesions such as 8-oxoG \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Here, we show that the protective LoL-DDR is dysregulated in transformed cell lines. 1-RIR still accumulate under high replication stress conditions, which differs from RIR levels in primary cells; 2- cellular RIR production occurs independently of NADPH oxidase control; and 3- the accumulation of premutagenic 8-oxoG in the cell genome. In addition, experiments using an \u003cem\u003ein vitro\u003c/em\u003e cellular model of breast cancer progression suggested that dysregulation of the LoL-DDR occurs in an early stage of cancer progression.\u003c/p\u003e \u003cp\u003eAn increase in premutagenic lesions with increased replication stress should induce mutagenesis, resulting in genetic instability that could foster cancer initiation and progression as well as the appearance of neoantigens. Importantly, the process described here has been shown to occur in cell lines with wild-type p53 (RKO, U2OS, and RPE-tert cells) as well as in cells with mutated p53 (V79 cells) or in which p53 is trapped by the SV40 large T antigen (RG37 cells). Thus RIR production in immortalized/transformed cell lines occurs independently of p53. These data are consistent with the fact that LoL-DDR dysregulation occurs in an early stage of cancer progression, as shown here.\u003c/p\u003e \u003cp\u003eAs summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, in primary noncancer cells, nonblocking, low-level replication stress induces the LoL-DDR, which protects genome stability; increasing the stress intensity turns off the LoL-DDR but induces the cDDR, which maintains genetic stability but in a different way. Notably, all the factors that play pivotal roles in controlling the LoL-DDR, are lost in different types of tumors \u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30 CR31 CR32 CR33\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In immortalized cells, the LoL-DDR is dysregulated in the early stage of cancer progression, and replication stress induces ROS production even at high levels of stress, leading to the accumulation of premutagenic lesions in the genome. Since, ROS cause replication stress \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, this triggers a vicious cycle, which maintains and amplifies genetic instability and thus should play an important role in cancer etiology in the both initiation and progression stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and treatments\u003c/h2\u003e \u003cp\u003eThe cells were grown at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in DMEM supplemented with 10% fetal calf serum (FCS).\u003c/p\u003e \u003cp\u003ePrimary human mammary epithelial cells (HuMECs) were obtained from Thermo Fisher Scientific and cultured according to the manufacturer\u0026rsquo;s recommendations. HuMEC derivatives (HME, HMLE, and HMLER cells) were obtained from Professors Alain Puisieux (Lyon, France) and Robert Weinberg (Boston, USA) and were cultured in 1:1 DMEM/HAM-F12 medium (Gibco, Life Technologies) supplemented with 10% FBS (Lonza Group, Ltd.), 100 U/ml penicillin‒streptomycin (Invitrogen), 10 ng/ml human epidermal growth factor (EGF) (Sigma), 0.5 \u0026micro;g/ml hydrocortisone (Sigma) and 10 \u0026micro;g/ml insulin (Sigma). Primary fibroblasts were exposed to HU, APH or CPT for 72 h at 37\u0026deg;C. For antioxidant treatment, primary fibroblasts were exposed to 2 mM NAC (Sigma‒Aldrich, St. Louis, MO, USA) for 72 h.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMeasurement of cellular ROS production by FACS analysis.\u003c/b\u003e Cellular ROS production was measured with a CM-H2DCFDA (2\u0026rsquo;,7\u0026rsquo;-dichlorofluorescein diacetate) (Life Technologies, USA) or dihydrorhodamine 123 (DHR 123) (Sigma) assay kit according to the manufacturer\u0026rsquo;s protocol. Approximately 10\u003csup\u003e5\u003c/sup\u003e cells/well were plated into 6-well plates and incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. After 2 days, the cells were washed with PBS and incubated with 10 \u0026micro;M CM-H2DCFDA or DHR 123 in DMEM supplemented with 1% FBS for 45 min at 37\u0026deg;C in the dark. The cells were trypsinized and resuspended in DMEM supplemented with 1% FBS. The pelleted cells were washed again, and the live cells were resuspended in PBS and analyzed on a BD Accuri C6 flow cytometer (BD Biosciences, San Diego, CA) equipped with an FL1 laser (515\u0026ndash;545 nm). The data are presented as the mean percentages of four independent experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell cycle analysis and BrdU incorporation.\u003c/b\u003e Cells were incubated in culture medium supplemented with or without HU for 72 h at 37\u0026deg;C, and 5-bromo-2-deoxyuridine (BrdU, Sigma) was added to the culture medium at a final concentration of 10 \u0026micro;M and incubated for 30 min. Pelleted cells were detached with trypsin, fixed with 80% ethanol, and resuspended in 30 mM HCl/0.5 mg/ml pepsin. BrdU was immunofluorescently labeled with a mouse anti-BrdU antibody (DAKO, clone Bu20a) and a fluorescein-conjugated donkey anti-mouse antibody (Life Technologies), and the cells were stained with propidium iodide (PI; 25 \u0026micro;g/ml) in the presence of ribonuclease A (50 \u0026micro;g/ml). Flow cytometry analyses were performed using an Accuri C6 flow cytometer (BD Biosciences).\u003c/p\u003e \u003cp\u003e \u003cb\u003e8-OxoG measurement.\u003c/b\u003e Genomic DNA was extracted and enzymatically digested using an optimized protocol that minimizes DNA oxidation during the procedure \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Then, 8-oxoG levels were quantified using isotope dilution high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-MS/MS) as previously described \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e; \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN\u003csub\u003e5\u003c/sub\u003e-8-oxoG was used as the internal standard. In addition to the mass spectrometric detector, the system is equipped with a UV detector that is set to 260 nm to measure the quantity of normal nucleosides. The results are expressed as the number of 8-oxoG lesions per million normal nucleosides.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analyses.\u003c/b\u003e Statistical analyses were performed with GraphPad Prism 10. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS.\u0026nbsp;\u003c/strong\u003eWe thank Corinne Dupuy (Gustave Roussy Cancer Institute) for helpful discussions and advice on NADPH oxidases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions.\u0026nbsp;\u003c/strong\u003eConceptualization: BSL; funding acquisition: BSL; investigation: SR, ED, SC; methodology: SR, BSL, SC, and J-LR; 8-oxoG dosage experiments: SC and J-L R; project administration: BSL; resources: BSL; supervision: BSL; validation: SR, ED, J-LR, and BSL; writing the original draft: BSL; and writing \u0026ndash; reviewing \u0026amp; editing: all the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability.\u0026nbsp;\u003c/strong\u003eAll data generated or analysed during this study are included in this published article. If needed, more details or explanations can be available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interests.\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was supported by grants from the Institut National du Cancer (PLBIO21-072, PLBIO24-194), La Ligue Nationale Contre Le Cancer (ARN th\u0026eacute;rapeutiques), Ligue r\u0026eacute;gionale contre le cancer (comit\u0026eacute; Ile de France), (ITMO Cancer (PCSI 2022), and Fondation ARC (ARCPJA2022060005157).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJackson, S. P. \u0026amp; Bartek, J. 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However, unchallenged cells proliferate when they are continually exposed to low-level/endogenous replication stress. We previously characterized a noncanonical response specific to nonblocking replication stress, i.e. low-level stress (LoL-DDR), in primary cells. Although LoL-DDR generates replication stress-induced ROS (RIR), it prevents the accumulation of premutagenic 8-oxo-guanine (8-oxoG). Primary cells control RIR production via NADPH oxidases. Increasing the severity of replication stress above a threshold triggers the cDDR, leading to cell cycle arrest and RIR suppression, resulting in a peak-shaped dose response for RIR production. Here, we show that the LoL-DDR is dysregulated in cancer cell lines, which exhibit the following differences compared with primary cells: 1- RIR are not detoxified under high-level stress, resulting in a continuous increase in the dose‒response curve of RIR production; 2- RIR are not produced by NADPH oxidases; 3- replication stress favors the accumulation of the premutagenic 8-oxoG. Moreover, using an \u003cem\u003ein vitro\u003c/em\u003e breast cancer progression model, we show that LoL-DDR dysregulation occurs at an early stage of cancer progression. Since, conversely, ROS trigger replication stress this establishes a \u0026ldquo;vicious circle\u0026rdquo; replication-stress/ROS that continuously jeopardizes genome integrity that should fuel and amplify tumorigenesis.\u003c/p\u003e","manuscriptTitle":"Dysregulation of the low-level replication stress response in transformed cell lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-14 10:15:32","doi":"10.21203/rs.3.rs-6155647/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-11T10:46:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-10T17:36:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-01T01:02:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"183680914826084015882053614431557941256","date":"2025-03-23T15:41:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199684987955345240239288166474439251797","date":"2025-03-21T08:43:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-20T03:56:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-20T03:33:30+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-12T13:44:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-12T10:08:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-04T15:34:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"01877fb3-0e33-47b9-bcb6-3e23b30c4b69","owner":[],"postedDate":"March 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45585312,"name":"Biological sciences/Molecular biology"},{"id":45585313,"name":"Biological sciences/Molecular biology/Dna damage and repair"},{"id":45585314,"name":"Biological sciences/Molecular biology/Dna damage and repair/Dna damage response"}],"tags":[],"updatedAt":"2025-06-09T16:04:32+00:00","versionOfRecord":{"articleIdentity":"rs-6155647","link":"https://doi.org/10.1038/s41598-025-05172-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-06-06 15:57:05","publishedOnDateReadable":"June 6th, 2025"},"versionCreatedAt":"2025-03-14 10:15:32","video":"","vorDoi":"10.1038/s41598-025-05172-0","vorDoiUrl":"https://doi.org/10.1038/s41598-025-05172-0","workflowStages":[]},"version":"v1","identity":"rs-6155647","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6155647","identity":"rs-6155647","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}