PO.ET02.07 · 实验与分子治疗
绘制细胞命运图谱:从代谢动态到DNA损伤反应
Mapping cellular fate: From metabolic dynamics to DNA damage responses
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
细胞稳态通过代谢活动、细胞周期进程、程序性细胞死亡和基因组完整性之间受到严格调控的相互作用来维持。这些通路的扰动是肿瘤发生的一个标志。对这些过程的分析不仅阐明了疾病机制,还为治疗策略提供了信息。在此,我们提出一个一体化的实验框架,旨在探究永生化细胞系和人原代细胞中的代谢、细胞周期调控、凋亡和DNA损伤反应(DDR)。为评估细胞的代谢依赖性,我们采用了SCENITH方法[1],该方法以嘌呤霉素掺入作为翻译活性(进而作为ATP产生)的替代指标。该方法能够在特定条件下定量细胞对糖酵解与氧化磷酸化的依赖程度。使用代谢抑制剂——2-脱氧葡萄糖阻断糖酵解、寡霉素抑制线粒体ATP合酶——我们观察到不同的代谢特征。CD8+ T细胞表现出对氧化磷酸化的明显依赖,而HeLa细胞尽管线粒体受到抑制仍维持翻译活性,提示其代谢灵活性。在进行代谢特征分析的同时,我们采用FxCycle™染色的流式细胞术细胞周期分析,以测定DNA含量和细胞的增殖状态。将细胞系MOLT-4暴露于具有确立抗增殖活性的疗法中。顺铂和帕博西尼(palbociclib)分别诱导G1期阻滞,分别与DNA交联和CDK4/6抑制一致,而微管破坏剂诺考达唑(nocodazole)使细胞阻滞于G2/M期。同时通过caspase 3/DAPI染色评估凋亡反应,结果显示与帕博西尼和诺考达唑相比,顺铂是最强效的早期和晚期凋亡诱导剂。最后,通过监测顺铂处理或辐照暴露后gammaH2AX和RAD51焦点的形成来定量DDR。高内涵成像有助于剂量反应特征分析,并证明与共济失调-毛细血管扩张症突变(ATM)激酶抑制剂联合处理显著放大了DNA损伤的累积,凸显了DDR调控的治疗潜力。总之,这些试验提供了一个可定制的平台,用于剖析药理学应激下的细胞脆弱性和韧性。此类多维特征分析为精准肿瘤学和免疫治疗提供了关键见解,能够识别情境特异性靶点和克服耐药的组合策略。[1] Arguello 2020 https://pmc.ncbi.nlm.nih.gov/articles/PMC8407169/#ABS1
查看英文原文 English abstract
Cellular homeostasis is maintained through a tightly regulated interplay of metabolic activity, cell cycle progression, programmed cell death, and genomic integrity. Perturbation of these pathways is a hallmark of oncogenesis. Analysis of these processes not only elucidates mechanisms of disease but also informs on therapeutic strategies. Here we present an integrated experimental framework designed to interrogate metabolism, cell cycle regulation, apoptosis, and DNA damage responses (DDR) in both immortalized cell lines and human primary cells. To assess cellular metabolic dependencies, we employed the SCENITH approach [1], which uses puromycin incorporation as a proxy for translational activity and, by extension, ATP production. This method enables quantification of reliance on glycolysis versus oxidative phosphorylation under defined conditions. Using metabolic inhibitors; 2-deoxyglucose to block glycolysis and oligomycin to inhibit mitochondrial ATP synthase, we observed distinct metabolic profiles. CD8⁺ T cells exhibited pronounced dependence on oxidative phosphorylation, whereas HeLa cells maintained translational activity despite mitochondrial inhibition, indicating metabolic flexibility. Parallel to metabolic profiling, we implemented flow cytometric cell cycle analysis using FxCycle™ staining to determine DNA content and the proliferative state of cells. Cell lines, MOLT-4, were exposed to therapeutics with established antiproliferative activity. Cisplatin and palbociclib induced G₁-phase arrest, consistent with DNA crosslinking and CDK4/6 inhibition, respectively, while nocodazole, a microtubule disruptor, arrested cells in G₂/M. Apoptotic responses were concurrently evaluated via caspase 3 /DAPI staining, revealing cisplatin as the most potent inducer of early and late apoptosis compared to palbociclib and nocodazole. Finally, DDR was quantified by monitoring gammaH2AX and RAD51 foci formation following cisplatin treatment or exposure to irradiation. High-content imaging facilitated dose-response characterization and demonstrated that co-treatment with an ataxia-telangiectasia mutated (ATM) kinase inhibitor markedly amplified DNA damage accumulation, underscoring the therapeutic potential of DDR modulation. Collectively, these assays provide a customizable platform for dissecting cellular vulnerabilities and resilience under pharmacological stress. Such multidimensional profiling offers critical insights for precision oncology and immunotherapy, enabling the identification of context-specific targets and combinatorial strategies to overcome resistance. [1] Arguello 2020 https://pmc.ncbi.nlm.nih.gov/articles/PMC8407169/#ABS1
利益披露 Disclosure
L. Sueiro-Ballesteros, None..
H. Leonard, None..
D. Rocca, None..
L. Schewitz-Bowers, None..
L. Brackenbury, None.