The Possible Role of Mitochondrial ATM, CDK-1, and RAN Expression in the Radioresistance of Human Lung Adenocarcinoma A549 Cells

The Possible Role of Mitochondrial ATM, CDK-1, and RAN Expression in the Radioresistance of Human Lung Adenocarcinoma A549 Cells | 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 Research Article The Possible Role of Mitochondrial ATM, CDK-1, and RAN Expression in the Radioresistance of Human Lung Adenocarcinoma A549 Cells Kemal Özbilgin, Mehmet Akif Can Akçalı, Kemal Atmaca, Yusuf Pekmezci, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8424369/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background/aim: Radiotherapy (RT) is a widely used treatment for non-small cell lung cancer (NSCLC), one of the most prevalent cancers globally. However, radioresistance (RR) remains a significant challenge, often leading to RT failure. The primary driver of RR is the enhanced DNA repair capability of tumor cells. Understanding the mechanisms underlying RR in NSCLC is essential for developing more effective therapeutic strategies. The ataxia-telangiectasia mutated (ATM) protein kinase is pivotal in responding to DNA double-strand breaks caused by RT. Additionally, cyclin-dependent kinase 1 (CDK1) and Ras-related nuclear protein (RAN) are crucial regulators of mitotic entry, G2 arrest, DNA repair, and cell cycle progression. Although these proteins are present in both cytosolic and mitochondrial forms, their specific roles in radiation response and RR remain poorly understood. Materials and methods: In this study, A549 cells were exposed to a single 5 Gy dose of ionizing radiation (IR) and analyzed at baseline and on days 1, 3, and 5 post-IR. The expression levels of ATM, CDK1, and RAN in cytosolic and mitochondrial fractions were assessed using immunohistochemical and Western blot analyses. Results: In immunohistochemical evaluations, ATM, CDK1, and RAN immunoreactivities A549 cancer cells were found to increase markedly on the 1st day, decreased on the 3rd day, and approached control group on the 5th day. Mitochondrial expressions of all three proteins increased significantly on day 1 but declined markedly by day 5. Conversely, altered cytosolic expressions returned to baseline levels by day 5. Conclusion: These results suggest that the early upregulation of mitochondrial ATM, CDK1, and RAN contributes to adaptive RR in A549 cancer cells, offering potential targets for overcoming RT resistance. A549 cells radiotherapy mitochondria ATM CDK1 RAN Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Background Non-small cell lung cancer (NSCLC) remains a significant global health challenge. Its treatment typically involves surgery, chemotherapy, immunotherapy, targeted therapy, and radiotherapy (RT). However, radio resistance (RR) is reported to induce resistance mechanisms in cancer cells, reducing the effectiveness of antitumor treatments (Zhou et al., 2003). The mechanisms underlying RR in NSCLC are complex and can be classified into inherent RR and acquired RR. Inherent RR is primarily associated with oncogenic mutations, cancer stem cells, and tumor hypoxia (Johung et al., 2013 ). In contrast, acquired RR is predominantly related to DNA damage and involves a process where cancer cells adapt to ionizing radiation (IR), ultimately leading to treatment resistance (Porrazzo et al., 2024 ). RR contributes to cancer relapse, poor treatment response, unfavorable prognosis, decreased quality of life, and an increased burden of disease management. IR induces direct DNA damage, such as single-strand breaks (SSBs) and double-strand breaks (DSBs), as well as indirect damage through oxidative stress caused by reactive oxygen and reactive nitrogen species. Among these, DSBs generated by RT are the most effective molecular events for damaging and killing cancer cells. Genes and proteins involved in DSB repair are critical targets for cancer therapy, as their alteration, interaction, translocation, or regulation can significantly affect the repair process, influencing cancer cell sensitivity or resistance to radiotherapy (Mladenov et al., 2013 ). The rapid natural DNA damage repair mechanisms in cancer cells are considered a primary factor contributing to RR (O'Connor et al., 2015). IR induces replication-independent DSBs that can kill non-replicating cells. It also rapidly inhibits DNA replication by activating cell cycle checkpoints to prevent the formation of toxic DNA replication lesions (Painter et al., 1967). Cell cycle checkpoints are primarily regulated by the ataxia-telangiectasia mutated (ATM) gene by phosphorylation of numerous downstream effectors (Falck et al., 2001 ). ATM binds DNA break sites via the MRN complex, phosphorylates γH2AX, and initiates DNA repair (Carrassa et al., 2017 8). Specifically: the ATM-p53-p21 and ATM-Chk2-CDC25A pathways control G1/S arrest, the ATM-BRCA1/FANCD2/NBS1/SMC1 pathways regulate S-phase arrest, the ATM-Chk2-CDC25C, ATM-BRCA1-cyclin B, and ATM-p53-CDC2-cyclin B1 pathways govern G2/M phase arrest (Carrassa et al., 2017). Inhibiting ATM function could sensitize tumor cells to IR and chemotherapy, potentially improving the treatment response in patients with chemoresistance and RR. Cyclin-dependent kinase 1 (CDK1) is essential for cell cycle regulation, including the centrosome cycle, mitotic onset, G2/M transition, and G1/S transition in combination with different cyclins (Wang et al., 2018 ). CDK1 activation involves multiple steps; CDK1 binds to cyclin B, phosphorylation of Thr161 is induced by the CAK/CDK7 complex, phosphorylation at Thr14 and Tyr15 is inhibited by CDC25, and the CDK1/cyclin B complex is activated, redirecting cyclin-kinase complexes from the cytoplasm to the nucleus (Yu et al., 2019 ). During DNA damage caused by IR, CDK1 is inactivated to halt the cell cycle at the G1, S, or G2 checkpoints, facilitating DSB repair (Wang et al., 2015 ). Given that cancer cells rely on specific interphase CDKs for proliferation, inhibition/downregulation of CDK1 could offer therapeutic benefits against RR. Ras-related nuclear protein (RAN) is an essential regulator of nucleocytoplasmic transport during interphase and plays a critical role in mitosis by controlling DNA synthesis and cell cycle progression (Nevo et al., 2003 , Scheffzek et al., 1995 ). Mutations in RAN have been shown to disrupt DNA synthesis, further supporting its role in this process (Sazer et al., 2000). In addition to its primary function in nucleocytoplasmic transport, RAN regulates the formation and organization of the microtubule network in a transport-independent manner (Ren et al.1994, Wilde et al.2001, Bamba et al.,2002). This regulatory role is significant for processes such as spindle assembly, chromosome positioning, and nuclear envelope formation. RAN also functions as an androgen receptor coactivator and has been implicated in various diseases (Sampson et al., 2001 ). Given its diverse intracellular roles, RAN likely interacts with multiple proteins to execute its functions. Considering the closely related roles of RAN, ATM, and CDK1 in cell cycle regulation and DNA synthesis, an important question remains: how is RAN modulated by IR. In recent years, mitochondria have been suggested to play a role in RR, though the exact mechanisms remain unclear. Known as the cell’s powerhouse, mitochondria are crucial for energy production and regulate various cellular processes, including energy metabolism, apoptosis, reactive oxygen species (ROS) generation, and cancer development and progression (Bian et al., 2022 ). Mitochondria contribute to cancer by influencing cell growth, evasion of cell death, and metastasis (Zampieri et al., 2021 ). Radiation can damage mitochondrial DNA (mtDNA), which resides in the mitochondrial matrix and lacks robust self-repair mechanisms (Wang et al., 2017 .) Damaged mtDNA can lead to mitochondrial dysfunction, and alterations such as mutations, deletions, or variations in mtDNA copy number may significantly impact a cell’s response to radiation (van Gisbergen et al., 2017 ). Mitochondria also regulate cell death pathways, and dysfunctional mitochondria have been linked to heightened RR in cancer cells. Structural abnormalities and defects in mitochondria strongly correlate with malignancy and RR (Cloos et al., 2009 ). Moreover, numerous studies suggest that mitochondrial metabolism plays a central role in the development of RR. This connection arises not only from mitochondria’s involvement in radiation-induced regulated cell death tolerance but also from their role as key drivers of metabolic reprogramming. In this study, we aimed to investigate whether ATM, CDK1, and RAN are expressed in response to IR in both cytosolic and mitochondrial fractions of non-small cell lung cancer (NSCLC) cells, which are known to exhibit RR. Our findings provide novel insights into the mechanisms of action of a single dose of 5 Gy ionizing radiation in NSCLC cells and may contribute to strategies for preventing the development of RR. 2. Methods Cell Culture Since this study included only cell culture applications and a ready-made cell line was used, there is no ethical approval requirement. The materials used in the experiment were provided by the project unit of our university. The human non-small cell lung cancer (NSCLC) A549 cell line (CCL-185) was obtained from the American Type Culture Collection. Cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Biosera, USA), 100 U/ml penicillin, 100 µg/ml streptomycin (Biosera, USA), and 2 mM L-glutamate (Biosera, USA). The morphology and proliferation of the cells were observed daily under an inverted microscope. Cells were routinely monitored for mycoplasma contamination and maintained in a humidified incubator at 37°C with 5% CO₂. Subculturing was performed when cell confluence reached 80–90%. 2.1 Immunocytochemical Staining The control group remained untreated, while three experimental groups were exposed to a 5 Gy dose of IR for 2 minutes. Treated samples were fixed at 24 hours, 72 hours, and 120 hours post-exposure. In vitro radiotherapy was performed using a 4 MeV electron beam linear accelerator (Elekta, Stockholm, Sweden). Radiation dose applied in this study same as a study cited in Kurtman et al. ( 2022 ). Following treatment, all experimental group cells were fixed with 4% paraformaldehyde for 20 minutes, washed twice with PBS (5 minutes each), and permeabilized on ice for 15 minutes using 0.1% Triton X-100 (Applichem, 4L003808). Cells were then washed with PBS and incubated with 3% H₂O₂ (Merck, K31355100303) at room temperature for 10 minutes to block endogenous peroxidase activity. Cells were incubated in blocking solution (Invitrogen, 859043) for 1 hour at room temperature. Without washing, the blocking solution was removed, and primary antibodies -ATM (603652, BioLegend), Caspase-3 (bs-0081R, Bioss), CDK1 (NB100–2280, Novus Biologicals), and RAN (Boster Bio, A00204-1) were added at a 1:100 dilution. The samples were incubated overnight at 4°C. The next day, primary antibodies were removed by washing with PBS, and a biotinylated secondary antibody was applied for 30 minutes. Slides were then incubated with streptavidin (Invitrogen, 859043) for 30 minutes at room temperature. After washing, cells were treated with 3,3’-Diaminobenzidine (DAB) (AEM080, ScyTek Laboratories) for 5 minutes to visualize staining. Counterstaining was performed using Mayer’s Hematoxylin (72804E, Microm, Walldorf). All samples were observed under a light microscope (BX-40; Olympus). Quantitative analysis of immunohistochemical staining was conducted using Fiji software, as described by Patera et al. (23,24)). Optical density (mean gray value) was calculated using the formula: Optical Density = log (Maximum Gray Density/Average Gray Density) . The quantification of immunoreactivity was performed using ImageJ software with the Colour Deconvolution2 plugin ( https://github.com/landinig/IJ-Colour_Deconvolution2?tab=readme-ov-file ). In relative scoring, each 10X microscopic image was analyzed separately for DAB and hematoxylin staining. The pixel counts for each staining type were calculated as percentages, and the final scores were expressed as the ratio of DAB to hematoxylin percentage. Statistical analysis of intergroup differences was conducted using non-parametric variance analysis in GraphPad Prism 9 software, with p < 0.05 considered statistically significant. 2.2Isolation of Mitochondria from Cells Half of the cells in each group were allocated for mitochondrial isolation. Approximately 1 × 10⁶ cells were resuspended in 250 µL of sucrose-mannitol buffer (20 mM HEPES, pH 7.5; 70 mM sucrose; 220 mM mannitol; 2 mM EDTA; 0.5 mg/mL BSA) and homogenized with 10–15 strokes using a glass potter. The homogenate was centrifuged at 600 g for 10 minutes at 4°C, followed by a second centrifugation at 650 g for 10 minutes to remove debris. The supernatant was then centrifuged at 8,000 g for 15 minutes to obtain the crude mitochondrial pellet. The resulting supernatant was separated to isolate the cytosolic fraction. The mitochondrial pellet was washed twice with sucrose-mannitol buffer and collected as the final mitochondrial fraction (Arzuk et al., 2000). 2.3 Preparation of Mitochondrial Homogenate The mitochondrial pellet was resuspended in 100 µL of RIPA buffer and lysed in an ice bath using an ultrasonicator set at 60 kHz for 1 minute. The resulting homogenate was kept on ice for 10 minutes. After incubation, the homogenate was centrifuged at 14,000 g at 4°C for 5 minutes. The supernatant was transferred to clean tubes and stored at -80°C until further analysis. 2.4Western Blot (WB) Analysis of Cytosolic and Mitochondrial Fractions Protein content in the homogenates was quantified using the Bradford assay. Proteins from mitochondrial and cytosolic fractions (15 µg each) were loaded onto 12% polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoresed in running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at 120 V until protein bands separated. Subsequently, the proteins were transferred to PVDF membranes at 120 V for 120 minutes in a transfer buffer (20% w/v methanol, 0.025% w/v SDS, 48 mM Tris, 39 mM glycine) at 4°C. Following transfer, the membranes were blocked for 1 hour in 5% nonfat dry milk prepared in Tris-buffered saline containing 0.1% Tween 20 (TBST) to prevent non-specific binding. The membranes were treated with primary antibodies (anti-ATM, anti-CDK, anti-RAN, anti-HSP60, and anti-GAPDH), washed 3 times with TBS-T buffer, followed by incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (dilution: 1:3,000) for 2 hours at room temperature. The membranes were then washed again at least 3 times with TBS-T buffer and incubated for 5 min at room temperature using the ECL kit. They were placed on a Vilber Lourmat Fusion FX7 imaging system and protein bands were visualized using Clarity Western Blotting reagent (Mahmood et al., 2012). 3. Results 3.1 Immunocytochemical Staining Results In preparations of the human NSCLC A549 cell line stained with the ATM primary antibody, low levels of ATM immunoreactivity were observed in the control group. In the irradiated groups, ATM immunoreactivity increased significantly on day 1 after irradiation, but decreased markedly on day 3. On day 5, ATM immunoreactivity was increased as day 1 (Table 1 Fig. 1 ). CDK1 immunoreactivity was observed at low levels in the control group. CDK1 immunoreactivity increased on the 1st and 3rd days in the irradiated groups. However, the increased CDK1 immunoreactivity was observed to be more prominent on the 5th day (Table 1 Fig. 1 ). RAN immunoreactivity was similar in all groups. While a slight increase in RAN immunoreactivity was observed on the first day after radiation, this increase was not significant on days 3 and 5. (Table 1 Fig. 1 ). Table 1 The immunohistochemical staining intensity of ATM, CDK1 and RAN Control 1.Day 3.Day 5.Day ATM 0,0223 (±) 0,0189 0,2480 (±) 0,0939 0,0531 (±) 0,0071 0,2892 (±) 0,0796 CDK 0,0603 (±) 0,0355 0,1412 (±) 0,0542 0,1303 (±) 0,1282 0,3718 (±) 0,5341 RAN 0,0328 (±) 0,0119 0,0435 (±) 0,0223 0,0327 (±) 0,0232 0,0387 (±) 0,0267 3.2 Western Blot Results The expression of ATM in the cytosolic fractions of NSCLC A549 cell line samples was detected by Western blot (Fig. 1 ). ATM was significantly upregulated in the 1st-day samples from the irradiated groups compared to the control group. However, ATM protein expression in the cytosolic fractions decreased on the 3rd and 5th days post-radiation, returning to levels similar to those observed in the control group. When examining ATM protein expression in the mitochondrial fractions, a significant increase was observed on the 1st day post-radiation, mirroring the trend seen in the cytosol. Conversely, ATM expression in the mitochondrial fractions was significantly lower than that of the control group on the 3rd and 5th days post-radiation (Fig. 2 ). In the NSCLC A549 cell line, CDK1 expression in the cytosolic fractions was observed in the control group (Fig. 2 ). On the 1st day post-radiation, CDK1 expression was comparable to the control group. However, it significantly decreased on the 3rd and 5th days post-radiation. In the mitochondrial fractions of the control group samples, CDK1 expression was observed to be quite low compared to the cytosol. Notably, CDK1 expression in the mitochondrial fractions significantly increased on the 1st day post-radiation compared to the control group. By the 3rd and 5th days post-radiation, CDK1 expression in the mitochondrial fractions returned to levels similar to those in the control group and showed a significant decline compared to the 1st day post-radiation (Fig. 3 ). The response of both cytosolic and mitochondrial RAN to a single 5 Gy dose of ionizing radiation is presented in Fig. 4 . Cytosolic RAN levels remained unchanged on the 1st day post-radiation. However, they significantly decreased on the 3rd day and nearly returned to control levels with a slight increase by the 5th day (Fig. 4 ). In contrast, mitochondrial RAN exhibited a markedly different response to radiation. Its levels significantly increased on the 1st day post-radiation and remained elevated on the 3rd day. However, a significant decrease was observed by the 5th day, compared to both control and day 3 (Fig. 4 ). 4. Discussion RT is one of the most commonly used treatments for lung cancer. However, intrinsic and acquired RR of tumor cells remains a significant clinical obstacle, particularly in NSCLC. Increasing evidence highlights the role of mitochondrial functions as key modulators of radiosensitivity and contributors to RR in cancer cells. Consequently, investigating the impact of IR on mitochondrial functions is essential for understanding RR and developing therapeutic strategies to enhance RT efficacy in cancer patients. DNA repair capacity is a major determinant of RR in cancer cells (O'Connor et al.,2015). In this study, we observed an increased expression of ATM in the cytosolic fraction of A549 cells one day post-IR, suggesting its involvement in RR. Upon IR exposure, cytoplasmic ATM dimers monomerize and migrate to the nucleus (Maeda et al.,2021). DSBs in DNA rapidly activate ATM via autophosphorylation, which is critical for DNA damage repair (Kastan et al.,2000). Our findings showed increased ATM expression in both cytosolic and mitochondrial fractions of A549 cells on day 1 post-IR, which returned to control levels in the cytosol and decreased below control levels in mitochondria by day 5. These results suggest that the transient increase in ATM expression may facilitate homologous recombination and strengthen RR by enabling efficient repair of nuclear DNA damage. ATM plays a pivotal role in repairing approximately 15% of radiation-induced DSBs through mediator proteins such as MDC1 and 53BP1 (Goodarzi et al.,2010). Conversely, ATM deficiency sensitizes cells to IR by disrupting DNA repair, activating CHK2 kinase, and inducing apoptosis or cell cycle arrest (Hirao et al., 2002 ). In addition to its role in nuclear DNA repair, ATM contributes to the AMPK pathway following radiation, transmitting DNA damage signals to mitochondria to maintain genomic stability in irradiated cells (Shimura et al.,2024). The mitotic increase in ATM expression observed on day 1 in A549 cells may reflect its role in promoting genomic stability. Furthermore, AMPK enhances mitochondrial biogenesis through transcription factors such as NRF-1, NRF-2, and TFAM (Zhang et al., 2017). The increase in mitochondrial ATM expression observed on day 1 may be linked to mitochondrial biogenesis, as evidenced by a significant increase in mitochondrial mass following a 5 Gy IR dose (Atmaca et al., 2025, in press). ATM-deficient cells exhibit a radiosensitive phenotype, emphasizing its role in modulating mitochondrial homeostasis under genotoxic stress (Shimura et al.,2021). IR-induced mitochondrial oxidative phosphorylation supports the energy demands of DNA restructuring, damage signaling, and repair processes (Yamamori et al.,2012). However, ATM inhibition or deficiency impairs mitochondrial responses to radiation (Shimura et al.,2024). ATM has been shown to colocalize with PINK1 and Parkin, indicating its role in PINK1/Parkin-mediated mitophagy (Gu et al.,2018). Our data suggests that increased mitochondrial ATM expression on day 1 post-IR may facilitate selective elimination of damaged mitochondria through mitophagy. Cyclin B1/CDK1 expression is induced to arrest the cell cycle at G2/M under genomic stress, allowing time for DNA repair or initiating apoptosis (Sherwood et al.,1994). Our study revealed distinct responses of cytosolic and mitochondrial CDK1 to IR: cytosolic CDK1 decreased significantly on day 3 and was restored by day 5, while mitochondrial CDK1 increased dramatically on day 1 and decreased progressively by day 5. This differential expression pattern suggests that cytosolic CDK1 regulates cell cycle progression (Sriramulu et al.,2023, Wang et al., 2018 ), whereas mitochondrial CDK1 may play a role in mitochondrial dynamics and RR. Mitochondrial Cyclin B1/CDK1 activity has been implicated in mitochondrial biogenesis, energy production, and resistance to IR (Xie et al.,2018). Our findings align with studies reporting that Cyclin B1/CDK1 enhances SIRT3 deacetylation activity, reducing mitochondrial protein acetylation and ATP generation, and influencing radiosensitivity (Liu et al., 2015 ). Conflicting reports indicate that inhibiting CDK1 can increase radiosensitivity, highlighting the complexity of its role (Wang et al., 2015 ). Mitochondria respond to IR by recruiting Cyclin B1/CDK1 to increase MnSOD activity, stabilize mitochondrial function, and support cellular adaptation (Candas et al.,2013). Our observation of increased mitochondrial CDK1 expression on day 1 post-IR suggests that it may contribute to cancer cell survival and RR. MnSOD activity exhibited distinct patterns in cancerous and healthy cells post-IR (Atmaca et al., 2025, in press), further supporting the adaptive role of mitochondrial responses in RR. Lastly, this study provides the first insights into the responses of cytosolic and mitochondrial RAN to a single 5 Gy IR dose. Cytosolic RAN expression decreased significantly on day 3, while mitochondrial RAN expression increased on day 1, remained elevated on day 3, and decreased significantly by day 5. Given the critical role of RAN in DNA synthesis (Sazer et al.,2000), the prompt increase in mitochondrial RAN suggests an adaptive response to maintain mtDNA integrity, which is more vulnerable to IR than nuclear DNA. In conclusion, this study demonstrated that ATM, CDK1, and RAN are differentially expressed in cytosolic and mitochondrial fractions of A549 cells post-IR. The transient increase in their mitochondrial expression on day 1 post-IR likely contributes to genomic stability and tumor RR by enhancing mitochondrial biogenesis. These findings identify potential therapeutic targets to overcome RT resistance and improve treatment outcomes. Abbreviations IR : ionizing radiation; RT : radiotherapy; RR : radioresistance; NSCLC : non-small cell lung cancer; ATM : ataxia-telangiectasia mutated protein kinase; CDK1 : Cyclin-dependent kinase 1; RAN : Ras-related nuclear protein; SSB : single-strand breaks; DSB : double-strand breaks; Declarations COMPETING INTERESTS The authors declares that they have no competing interests. Ethical Approval and Consent to Participate: Since this study is a cell culture study with a cell line, it does not need to obtain any ethical approval in our country. Consent for Publication: All authors have consented to the publication of the study. Funding: The study was funded by the scientific research project unit, which could not be specified in order not to impair anonymity. Author Contribution K.Ö. , M.A.C.A and H.O. wrote the main maniscuript textK.Ö. , M.A.C.A and Y.P. did Immunocytochemical StainingsK.A. and H.O Western Blot Analysis of Cytosolic and Mitochondrial Fractions G.B. , Ö.K. and C.K. did Radiation Applications and Dose DeterminationAll authors reviewed the manuscript. References Arzuk E, Karakuş F, Orhan H (2021). Bioactivation of clozapine by mitochondria of the murine heart: Possible cause of cardiotoxicity. Toxicology 447: 152628. doi: 10.1016/j.tox.2020.152628. Bamba C, Bobinnec Y, Fukuda M, Nishida E (2002). The GTPase Ran regulates chromosome positioning and nuclear envelope assembly in vivo. Current Biology 19;12(6):503-7.doi: 10.1016/s0960-9822(02)00741-8 Bian C, Zheng Z, Su J, Wang H, Chang S, Xin Y, et al. (2022). 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Mitochondrial Transfer in Cancer: A Comprehensive Review International Journal of Molecular Sciences 23;22(6):3245. doi: 10.3390/ijms22063245. Zhou T, Zhang LY, He JZ, Miao ZM, Li YY, Zhang YM, et al. (2023). Review: Mechanisms and perspective treatment of radioresistance in non-small cell lung cancer. Frontiers in immunology 14;14 1133899. doi: 10.3389/fimmu.2023.1133899. Yu TY, Garcia VE, Symington LS. (2019). CDK and Mec1/Tel1-catalyzed phosphorylation of Sae2 regulate different responses to DNA damage. Nucleic Acids Reseach 47: 11238–11249. DOI: 10.1093/nar/gkz814 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-8424369","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":571320839,"identity":"1e785857-851b-402f-8f98-7e8aa23d7779","order_by":0,"name":"Kemal Özbilgin","email":"","orcid":"","institution":"Manisa Celal Bayar University","correspondingAuthor":false,"prefix":"","firstName":"Kemal","middleName":"","lastName":"Özbilgin","suffix":""},{"id":571320843,"identity":"a75ae56d-746c-4890-9a0b-e17277532054","order_by":1,"name":"Mehmet Akif Can 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1","display":"","copyAsset":false,"role":"figure","size":664856,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analyses of cytosolic levels of ATM, CDK1 and RAN proteins following a single dose of 5 Gy ionizing radiation over a period of 5 days.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8424369/v1/9e055843cf541ecf338bac5b.png"},{"id":100364884,"identity":"724423bf-1f72-4ce4-aae5-2811a8621362","added_by":"auto","created_at":"2026-01-16 07:54:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":91636,"visible":true,"origin":"","legend":"\u003cp\u003eLevels of cellular ATM levels following a single dose of 5 Gy ionizing radiation over a period of 5 days. (A) Western blots of cytosolic ATM; (B) Western blots of mitochondrial ATM; (C) Comparable levels of cytosolic and mitochondrial levels of ATM; \u003cem\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/em\u003esignificantly different from its own control (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8424369/v1/0235c5f3c3e116da62023b8e.png"},{"id":100075804,"identity":"a67910d9-2265-4d75-84db-51b3b4d4e62b","added_by":"auto","created_at":"2026-01-12 17:16:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90248,"visible":true,"origin":"","legend":"\u003cp\u003eLevels of cellular CDK1 following a single dose of 5 Gy ionizing radiation over a period of 5 days. (A) Western blots of cytosolic CDK1; (B) Western blots of mitochondrial CDK1; (C) Comparable levels of cytosolic and mitochondrial levels of CDK1; \u003cem\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/em\u003esignificantly different from its own control (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8424369/v1/c47b695ba3ea1997cfffd252.png"},{"id":100075807,"identity":"ee41be77-8f88-4646-849d-7f6de8f830ba","added_by":"auto","created_at":"2026-01-12 17:16:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":79238,"visible":true,"origin":"","legend":"\u003cp\u003eLevels of cellular RAN following a single dose of 5 Gy ionizing radiation over a period of 5 days. (A) Western blots of cytosolic RAN; (B) Western blots of mitochondrial RAN; (C) Comparable levels of cytosolic and mitochondrial levels of RAN; \u003cem\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/em\u003esignificantly different from its own control (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8424369/v1/ead38bce7a3f98351d63319b.png"},{"id":100364920,"identity":"9f5a39fe-20d7-46e9-bd5b-7665f1795518","added_by":"auto","created_at":"2026-01-16 07:54:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29091,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Result section.\u003c/p\u003e","description":"","filename":"unnumber.png","url":"https://assets-eu.researchsquare.com/files/rs-8424369/v1/2a8a12ea92e6d2b63af0924a.png"},{"id":107224669,"identity":"65d07b65-fe41-4be1-968d-8ee16de023dc","added_by":"auto","created_at":"2026-04-18 15:54:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1153398,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8424369/v1/ba963f0f-15c3-43da-a356-b02b09df7175.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Possible Role of Mitochondrial ATM, CDK-1, and RAN Expression in the Radioresistance of Human Lung Adenocarcinoma A549 Cells","fulltext":[{"header":"1. Background","content":"\u003cp\u003eNon-small cell lung cancer (NSCLC) remains a significant global health challenge. Its treatment typically involves surgery, chemotherapy, immunotherapy, targeted therapy, and radiotherapy (RT). However, radio resistance (RR) is reported to induce resistance mechanisms in cancer cells, reducing the effectiveness of antitumor treatments (Zhou et al., 2003). The mechanisms underlying RR in NSCLC are complex and can be classified into inherent RR and acquired RR. Inherent RR is primarily associated with oncogenic mutations, cancer stem cells, and tumor hypoxia (Johung et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In contrast, acquired RR is predominantly related to DNA damage and involves a process where cancer cells adapt to ionizing radiation (IR), ultimately leading to treatment resistance (Porrazzo et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). RR contributes to cancer relapse, poor treatment response, unfavorable prognosis, decreased quality of life, and an increased burden of disease management. IR induces direct DNA damage, such as single-strand breaks (SSBs) and double-strand breaks (DSBs), as well as indirect damage through oxidative stress caused by reactive oxygen and reactive nitrogen species. Among these, DSBs generated by RT are the most effective molecular events for damaging and killing cancer cells. Genes and proteins involved in DSB repair are critical targets for cancer therapy, as their alteration, interaction, translocation, or regulation can significantly affect the repair process, influencing cancer cell sensitivity or resistance to radiotherapy (Mladenov et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The rapid natural DNA damage repair mechanisms in cancer cells are considered a primary factor contributing to RR (O'Connor et al., 2015).\u003c/p\u003e \u003cp\u003eIR induces replication-independent DSBs that can kill non-replicating cells. It also rapidly inhibits DNA replication by activating cell cycle checkpoints to prevent the formation of toxic DNA replication lesions (Painter et al., 1967). Cell cycle checkpoints are primarily regulated by the ataxia-telangiectasia mutated (ATM) gene by phosphorylation of numerous downstream effectors (Falck et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). ATM binds DNA break sites via the MRN complex, phosphorylates γH2AX, and initiates DNA repair (Carrassa et al., 2017 8). Specifically: the ATM-p53-p21 and ATM-Chk2-CDC25A pathways control G1/S arrest, the ATM-BRCA1/FANCD2/NBS1/SMC1 pathways regulate S-phase arrest, the ATM-Chk2-CDC25C, ATM-BRCA1-cyclin B, and ATM-p53-CDC2-cyclin B1 pathways govern G2/M phase arrest (Carrassa et al., 2017). Inhibiting ATM function could sensitize tumor cells to IR and chemotherapy, potentially improving the treatment response in patients with chemoresistance and RR.\u003c/p\u003e \u003cp\u003eCyclin-dependent kinase 1 (CDK1) is essential for cell cycle regulation, including the centrosome cycle, mitotic onset, G2/M transition, and G1/S transition in combination with different cyclins (Wang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). CDK1 activation involves multiple steps; CDK1 binds to cyclin B, phosphorylation of Thr161 is induced by the CAK/CDK7 complex, phosphorylation at Thr14 and Tyr15 is inhibited by CDC25, and the CDK1/cyclin B complex is activated, redirecting cyclin-kinase complexes from the cytoplasm to the nucleus (Yu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). During DNA damage caused by IR, CDK1 is inactivated to halt the cell cycle at the G1, S, or G2 checkpoints, facilitating DSB repair (Wang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Given that cancer cells rely on specific interphase CDKs for proliferation, inhibition/downregulation of CDK1 could offer therapeutic benefits against RR.\u003c/p\u003e \u003cp\u003eRas-related nuclear protein (RAN) is an essential regulator of nucleocytoplasmic transport during interphase and plays a critical role in mitosis by controlling DNA synthesis and cell cycle progression (Nevo et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Scheffzek et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Mutations in RAN have been shown to disrupt DNA synthesis, further supporting its role in this process (Sazer et al., 2000). In addition to its primary function in nucleocytoplasmic transport, RAN regulates the formation and organization of the microtubule network in a transport-independent manner (Ren et al.1994, Wilde et al.2001, Bamba et al.,2002). This regulatory role is significant for processes such as spindle assembly, chromosome positioning, and nuclear envelope formation. RAN also functions as an androgen receptor coactivator and has been implicated in various diseases (Sampson et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Given its diverse intracellular roles, RAN likely interacts with multiple proteins to execute its functions. Considering the closely related roles of RAN, ATM, and CDK1 in cell cycle regulation and DNA synthesis, an important question remains: how is RAN modulated by IR.\u003c/p\u003e \u003cp\u003eIn recent years, mitochondria have been suggested to play a role in RR, though the exact mechanisms remain unclear. Known as the cell\u0026rsquo;s powerhouse, mitochondria are crucial for energy production and regulate various cellular processes, including energy metabolism, apoptosis, reactive oxygen species (ROS) generation, and cancer development and progression (Bian et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Mitochondria contribute to cancer by influencing cell growth, evasion of cell death, and metastasis (Zampieri et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Radiation can damage mitochondrial DNA (mtDNA), which resides in the mitochondrial matrix and lacks robust self-repair mechanisms (Wang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e.) Damaged mtDNA can lead to mitochondrial dysfunction, and alterations such as mutations, deletions, or variations in mtDNA copy number may significantly impact a cell\u0026rsquo;s response to radiation (van Gisbergen et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Mitochondria also regulate cell death pathways, and dysfunctional mitochondria have been linked to heightened RR in cancer cells. Structural abnormalities and defects in mitochondria strongly correlate with malignancy and RR (Cloos et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Moreover, numerous studies suggest that mitochondrial metabolism plays a central role in the development of RR. This connection arises not only from mitochondria\u0026rsquo;s involvement in radiation-induced regulated cell death tolerance but also from their role as key drivers of metabolic reprogramming.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to investigate whether ATM, CDK1, and RAN are expressed in response to IR in both cytosolic and mitochondrial fractions of non-small cell lung cancer (NSCLC) cells, which are known to exhibit RR. Our findings provide novel insights into the mechanisms of action of a single dose of 5 Gy ionizing radiation in NSCLC cells and may contribute to strategies for preventing the development of RR.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003e \u003cb\u003eCell Culture\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSince this study included only cell culture applications and a ready-made cell line was used, there is no ethical approval requirement. The materials used in the experiment were provided by the project unit of our university. The human non-small cell lung cancer (NSCLC) A549 cell line (CCL-185) was obtained from the American Type Culture Collection. Cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Biosera, USA), 100 U/ml penicillin, 100 \u0026micro;g/ml streptomycin (Biosera, USA), and 2 mM L-glutamate (Biosera, USA). The morphology and proliferation of the cells were observed daily under an inverted microscope. Cells were routinely monitored for mycoplasma contamination and maintained in a humidified incubator at 37\u0026deg;C with 5% CO₂. Subculturing was performed when cell confluence reached 80\u0026ndash;90%.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Immunocytochemical Staining\u003c/h2\u003e \u003cp\u003eThe control group remained untreated, while three experimental groups were exposed to a 5 Gy dose of IR for 2 minutes. Treated samples were fixed at 24 hours, 72 hours, and 120 hours post-exposure. In vitro radiotherapy was performed using a 4 MeV electron beam linear accelerator (Elekta, Stockholm, Sweden). Radiation dose applied in this study same as a study cited in Kurtman et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Following treatment, all experimental group cells were fixed with 4% paraformaldehyde for 20 minutes, washed twice with PBS (5 minutes each), and permeabilized on ice for 15 minutes using 0.1% Triton X-100 (Applichem, 4L003808). Cells were then washed with PBS and incubated with 3% H₂O₂ (Merck, K31355100303) at room temperature for 10 minutes to block endogenous peroxidase activity. Cells were incubated in blocking solution (Invitrogen, 859043) for 1 hour at room temperature. Without washing, the blocking solution was removed, and primary antibodies -ATM (603652, BioLegend), Caspase-3 (bs-0081R, Bioss), CDK1 (NB100\u0026ndash;2280, Novus Biologicals), and RAN (Boster Bio, A00204-1) were added at a 1:100 dilution. The samples were incubated overnight at 4\u0026deg;C. The next day, primary antibodies were removed by washing with PBS, and a biotinylated secondary antibody was applied for 30 minutes. Slides were then incubated with streptavidin (Invitrogen, 859043) for 30 minutes at room temperature. After washing, cells were treated with 3,3\u0026rsquo;-Diaminobenzidine (DAB) (AEM080, ScyTek Laboratories) for 5 minutes to visualize staining. Counterstaining was performed using Mayer\u0026rsquo;s Hematoxylin (72804E, Microm, Walldorf). All samples were observed under a light microscope (BX-40; Olympus). Quantitative analysis of immunohistochemical staining was conducted using Fiji software, as described by Patera et al. (23,24)). Optical density (mean gray value) was calculated using the formula: \u003cem\u003eOptical Density\u0026thinsp;=\u0026thinsp;log (Maximum Gray Density/Average Gray Density)\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe quantification of immunoreactivity was performed using ImageJ software with the Colour Deconvolution2 plugin (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/landinig/IJ-Colour_Deconvolution2?tab=readme-ov-file\u003c/span\u003e\u003cspan address=\"https://github.com/landinig/IJ-Colour_Deconvolution2?tab=readme-ov-file\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). In relative scoring, each 10X microscopic image was analyzed separately for DAB and hematoxylin staining. The pixel counts for each staining type were calculated as percentages, and the final scores were expressed as the ratio of DAB to hematoxylin percentage.\u003c/p\u003e \u003cp\u003eStatistical analysis of intergroup differences was conducted using non-parametric variance analysis in GraphPad Prism 9 software, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2Isolation of Mitochondria from Cells\u003c/h2\u003e \u003cp\u003eHalf of the cells in each group were allocated for mitochondrial isolation. Approximately 1 \u0026times; 10⁶ cells were resuspended in 250 \u0026micro;L of sucrose-mannitol buffer (20 mM HEPES, pH 7.5; 70 mM sucrose; 220 mM mannitol; 2 mM EDTA; 0.5 mg/mL BSA) and homogenized with 10\u0026ndash;15 strokes using a glass potter. The homogenate was centrifuged at 600 g for 10 minutes at 4\u0026deg;C, followed by a second centrifugation at 650 g for 10 minutes to remove debris. The supernatant was then centrifuged at 8,000 g for 15 minutes to obtain the crude mitochondrial pellet. The resulting supernatant was separated to isolate the cytosolic fraction. The mitochondrial pellet was washed twice with sucrose-mannitol buffer and collected as the final mitochondrial fraction (Arzuk et al., 2000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of Mitochondrial Homogenate\u003c/h2\u003e \u003cp\u003eThe mitochondrial pellet was resuspended in 100 \u0026micro;L of RIPA buffer and lysed in an ice bath using an ultrasonicator set at 60 kHz for 1 minute. The resulting homogenate was kept on ice for 10 minutes. After incubation, the homogenate was centrifuged at 14,000 g at 4\u0026deg;C for 5 minutes. The supernatant was transferred to clean tubes and stored at -80\u0026deg;C until further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4Western Blot (WB) Analysis of Cytosolic and Mitochondrial Fractions\u003c/h2\u003e \u003cp\u003eProtein content in the homogenates was quantified using the Bradford assay. Proteins from mitochondrial and cytosolic fractions (15 \u0026micro;g each) were loaded onto 12% polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoresed in running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at 120 V until protein bands separated. Subsequently, the proteins were transferred to PVDF membranes at 120 V for 120 minutes in a transfer buffer (20% w/v methanol, 0.025% w/v SDS, 48 mM Tris, 39 mM glycine) at 4\u0026deg;C. Following transfer, the membranes were blocked for 1 hour in 5% nonfat dry milk prepared in Tris-buffered saline containing 0.1% Tween 20 (TBST) to prevent non-specific binding. The membranes were treated with primary antibodies (anti-ATM, anti-CDK, anti-RAN, anti-HSP60, and anti-GAPDH), washed 3 times with TBS-T buffer, followed by incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (dilution: 1:3,000) for 2 hours at room temperature. The membranes were then washed again at least 3 times with TBS-T buffer and incubated for 5 min at room temperature using the ECL kit. They were placed on a Vilber Lourmat Fusion FX7 imaging system and protein bands were visualized using Clarity Western Blotting reagent (Mahmood et al., 2012).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Immunocytochemical Staining Results\u003c/h2\u003e \u003cp\u003eIn preparations of the human NSCLC A549 cell line stained with the ATM primary antibody, low levels of ATM immunoreactivity were observed in the control group. In the irradiated groups, ATM immunoreactivity increased significantly on day 1 after irradiation, but decreased markedly on day 3. On day 5, ATM immunoreactivity was increased as day 1 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCDK1 immunoreactivity was observed at low levels in the control group. CDK1 immunoreactivity increased on the 1st and 3rd days in the irradiated groups. However, the increased CDK1 immunoreactivity was observed to be more prominent on the 5th day (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRAN immunoreactivity was similar in all groups. While a slight increase in RAN immunoreactivity was observed on the first day after radiation, this increase was not significant on days 3 and 5. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe immunohistochemical staining intensity of ATM, CDK1 and RAN\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.Day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.Day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.Day\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0,0223 (\u0026plusmn;) 0,0189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0,2480 (\u0026plusmn;) 0,0939\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,0531 (\u0026plusmn;) 0,0071\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0,2892 (\u0026plusmn;) 0,0796\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCDK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0,0603 (\u0026plusmn;) 0,0355\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0,1412 (\u0026plusmn;) 0,0542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,1303 (\u0026plusmn;) 0,1282\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0,3718 (\u0026plusmn;) 0,5341\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0,0328 (\u0026plusmn;) 0,0119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0,0435 (\u0026plusmn;) 0,0223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,0327 (\u0026plusmn;) 0,0232\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0,0387 (\u0026plusmn;) 0,0267\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Western Blot Results\u003c/h2\u003e \u003cp\u003eThe expression of ATM in the cytosolic fractions of NSCLC A549 cell line samples was detected by Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). ATM was significantly upregulated in the 1st-day samples from the irradiated groups compared to the control group. However, ATM protein expression in the cytosolic fractions decreased on the 3rd and 5th days post-radiation, returning to levels similar to those observed in the control group. When examining ATM protein expression in the mitochondrial fractions, a significant increase was observed on the 1st day post-radiation, mirroring the trend seen in the cytosol. Conversely, ATM expression in the mitochondrial fractions was significantly lower than that of the control group on the 3rd and 5th days post-radiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the NSCLC A549 cell line, CDK1 expression in the cytosolic fractions was observed in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). On the 1st day post-radiation, CDK1 expression was comparable to the control group. However, it significantly decreased on the 3rd and 5th days post-radiation. In the mitochondrial fractions of the control group samples, CDK1 expression was observed to be quite low compared to the cytosol. Notably, CDK1 expression in the mitochondrial fractions significantly increased on the 1st day post-radiation compared to the control group. By the 3rd and 5th days post-radiation, CDK1 expression in the mitochondrial fractions returned to levels similar to those in the control group and showed a significant decline compared to the 1st day post-radiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe response of both cytosolic and mitochondrial RAN to a single 5 Gy dose of ionizing radiation is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Cytosolic RAN levels remained unchanged on the 1st day post-radiation. However, they significantly decreased on the 3rd day and nearly returned to control levels with a slight increase by the 5th day (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In contrast, mitochondrial RAN exhibited a markedly different response to radiation. Its levels significantly increased on the 1st day post-radiation and remained elevated on the 3rd day. However, a significant decrease was observed by the 5th day, compared to both control and day 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eRT is one of the most commonly used treatments for lung cancer. However, intrinsic and acquired RR of tumor cells remains a significant clinical obstacle, particularly in NSCLC. Increasing evidence highlights the role of mitochondrial functions as key modulators of radiosensitivity and contributors to RR in cancer cells. Consequently, investigating the impact of IR on mitochondrial functions is essential for understanding RR and developing therapeutic strategies to enhance RT efficacy in cancer patients.\u003c/p\u003e \u003cp\u003eDNA repair capacity is a major determinant of RR in cancer cells (O'Connor et al.,2015). In this study, we observed an increased expression of ATM in the cytosolic fraction of A549 cells one day post-IR, suggesting its involvement in RR. Upon IR exposure, cytoplasmic ATM dimers monomerize and migrate to the nucleus (Maeda et al.,2021). DSBs in DNA rapidly activate ATM via autophosphorylation, which is critical for DNA damage repair (Kastan et al.,2000). Our findings showed increased ATM expression in both cytosolic and mitochondrial fractions of A549 cells on day 1 post-IR, which returned to control levels in the cytosol and decreased below control levels in mitochondria by day 5. These results suggest that the transient increase in ATM expression may facilitate homologous recombination and strengthen RR by enabling efficient repair of nuclear DNA damage. ATM plays a pivotal role in repairing approximately 15% of radiation-induced DSBs through mediator proteins such as MDC1 and 53BP1 (Goodarzi et al.,2010). Conversely, ATM deficiency sensitizes cells to IR by disrupting DNA repair, activating CHK2 kinase, and inducing apoptosis or cell cycle arrest (Hirao et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to its role in nuclear DNA repair, ATM contributes to the AMPK pathway following radiation, transmitting DNA damage signals to mitochondria to maintain genomic stability in irradiated cells (Shimura et al.,2024). The mitotic increase in ATM expression observed on day 1 in A549 cells may reflect its role in promoting genomic stability. Furthermore, AMPK enhances mitochondrial biogenesis through transcription factors such as NRF-1, NRF-2, and TFAM (Zhang et al., 2017). The increase in mitochondrial ATM expression observed on day 1 may be linked to mitochondrial biogenesis, as evidenced by a significant increase in mitochondrial mass following a 5 Gy IR dose (Atmaca et al., 2025, in press). ATM-deficient cells exhibit a radiosensitive phenotype, emphasizing its role in modulating mitochondrial homeostasis under genotoxic stress (Shimura et al.,2021).\u003c/p\u003e \u003cp\u003eIR-induced mitochondrial oxidative phosphorylation supports the energy demands of DNA restructuring, damage signaling, and repair processes (Yamamori et al.,2012). However, ATM inhibition or deficiency impairs mitochondrial responses to radiation (Shimura et al.,2024). ATM has been shown to colocalize with PINK1 and Parkin, indicating its role in PINK1/Parkin-mediated mitophagy (Gu et al.,2018). Our data suggests that increased mitochondrial ATM expression on day 1 post-IR may facilitate selective elimination of damaged mitochondria through mitophagy.\u003c/p\u003e \u003cp\u003eCyclin B1/CDK1 expression is induced to arrest the cell cycle at G2/M under genomic stress, allowing time for DNA repair or initiating apoptosis (Sherwood et al.,1994). Our study revealed distinct responses of cytosolic and mitochondrial CDK1 to IR: cytosolic CDK1 decreased significantly on day 3 and was restored by day 5, while mitochondrial CDK1 increased dramatically on day 1 and decreased progressively by day 5. This differential expression pattern suggests that cytosolic CDK1 regulates cell cycle progression (Sriramulu et al.,2023, Wang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), whereas mitochondrial CDK1 may play a role in mitochondrial dynamics and RR. Mitochondrial Cyclin B1/CDK1 activity has been implicated in mitochondrial biogenesis, energy production, and resistance to IR (Xie et al.,2018). Our findings align with studies reporting that Cyclin B1/CDK1 enhances SIRT3 deacetylation activity, reducing mitochondrial protein acetylation and ATP generation, and influencing radiosensitivity (Liu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Conflicting reports indicate that inhibiting CDK1 can increase radiosensitivity, highlighting the complexity of its role (Wang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMitochondria respond to IR by recruiting Cyclin B1/CDK1 to increase MnSOD activity, stabilize mitochondrial function, and support cellular adaptation (Candas et al.,2013). Our observation of increased mitochondrial CDK1 expression on day 1 post-IR suggests that it may contribute to cancer cell survival and RR. MnSOD activity exhibited distinct patterns in cancerous and healthy cells post-IR (Atmaca et al., 2025, in press), further supporting the adaptive role of mitochondrial responses in RR.\u003c/p\u003e \u003cp\u003eLastly, this study provides the first insights into the responses of cytosolic and mitochondrial RAN to a single 5 Gy IR dose. Cytosolic RAN expression decreased significantly on day 3, while mitochondrial RAN expression increased on day 1, remained elevated on day 3, and decreased significantly by day 5. Given the critical role of RAN in DNA synthesis (Sazer et al.,2000), the prompt increase in mitochondrial RAN suggests an adaptive response to maintain mtDNA integrity, which is more vulnerable to IR than nuclear DNA.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrated that ATM, CDK1, and RAN are differentially expressed in cytosolic and mitochondrial fractions of A549 cells post-IR. The transient increase in their mitochondrial expression on day 1 post-IR likely contributes to genomic stability and tumor RR by enhancing mitochondrial biogenesis. These findings identify potential therapeutic targets to overcome RT resistance and improve treatment outcomes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eIR\u003c/strong\u003e: ionizing radiation; \u003cstrong\u003eRT\u003c/strong\u003e: radiotherapy; \u003cstrong\u003eRR\u003c/strong\u003e: radioresistance; \u003cstrong\u003eNSCLC\u003c/strong\u003e: non-small cell lung cancer; \u003cstrong\u003eATM\u003c/strong\u003e: ataxia-telangiectasia mutated protein kinase; \u003cstrong\u003eCDK1\u003c/strong\u003e: Cyclin-dependent kinase 1;\u0026nbsp;\u003cstrong\u003eRAN\u003c/strong\u003e: Ras-related nuclear protein; \u003cstrong\u003eSSB\u003c/strong\u003e: single-strand breaks; \u003cstrong\u003eDSB\u003c/strong\u003e: double-strand breaks;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declares that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to Participate:\u003c/strong\u003e Since this study is a cell culture study with a cell line, it does not need to obtain any ethical approval in our country.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication:\u003c/strong\u003e All authors have consented to the publication of the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The study was funded by the scientific research project unit, which could not be specified in order not to impair anonymity.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eK.\u0026Ouml;. , M.A.C.A and H.O. wrote the main maniscuript textK.\u0026Ouml;. , M.A.C.A and Y.P. did Immunocytochemical StainingsK.A. and H.O Western Blot Analysis of Cytosolic and Mitochondrial Fractions G.B. , \u0026Ouml;.K. and C.K. did Radiation Applications and Dose DeterminationAll authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eArzuk E, Karakuş F, Orhan H (2021). 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DOI: 10.1093/nar/gkz814\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"A549 cells, radiotherapy, mitochondria ATM, CDK1, RAN","lastPublishedDoi":"10.21203/rs.3.rs-8424369/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8424369/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground/aim: \u003c/strong\u003eRadiotherapy (RT) is a widely used treatment for non-small cell lung cancer (NSCLC), one of the most prevalent cancers globally. However, radioresistance (RR) remains a significant challenge, often leading to RT failure. The primary driver of RR is the enhanced DNA repair capability of tumor cells. Understanding the mechanisms underlying RR in NSCLC is essential for developing more effective therapeutic strategies. The ataxia-telangiectasia mutated (ATM) protein kinase is pivotal in responding to DNA double-strand breaks caused by RT. Additionally, cyclin-dependent kinase 1 (CDK1) and Ras-related nuclear protein (RAN) are crucial regulators of mitotic entry, G2 arrest, DNA repair, and cell cycle progression. Although these proteins are present in both cytosolic and mitochondrial forms, their specific roles in radiation response and RR remain poorly understood. Materials and methods: In this study, A549 cells were exposed to a single 5 Gy dose of ionizing radiation (IR) and analyzed at baseline and on days 1, 3, and 5 post-IR. The expression levels of ATM, CDK1, and RAN in cytosolic and mitochondrial fractions were assessed using immunohistochemical and Western blot analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eIn immunohistochemical evaluations, ATM, CDK1, and RAN immunoreactivities A549 cancer cells were found to increase markedly on the 1st day, decreased on the 3rd day, and approached control group on the 5th day. Mitochondrial expressions of all three proteins increased significantly on day 1 but declined markedly by day 5. Conversely, altered cytosolic expressions returned to baseline levels by day 5. Conclusion: These results suggest that the early upregulation of mitochondrial ATM, CDK1, and RAN contributes to adaptive RR in A549 cancer cells, offering potential targets for overcoming RT resistance.\u003c/p\u003e","manuscriptTitle":"The Possible Role of Mitochondrial ATM, CDK-1, and RAN Expression in the Radioresistance of Human Lung Adenocarcinoma A549 Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-12 17:16:24","doi":"10.21203/rs.3.rs-8424369/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c6151e04-9cb6-49f9-a2bf-71c498c20813","owner":[],"postedDate":"January 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-18T15:54:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-12 17:16:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8424369","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8424369","identity":"rs-8424369","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}