Influence of cell cycle on responses of MCF-7 cells to benzo[a]pyrene

BACKGROUND: Benzo[a]pyrene (BaP) is a widespread environmental genotoxic carcinogen that damages DNA by forming adducts. This damage along with activation of the aryl hydrocarbon receptor (AHR) induces complex transcriptional responses in cells. To investigate whether human cells are more susceptible to BaP in a particular phase of the cell cycle, synchronised breast carcinoma MCF-7 cells were exposed to BaP. Cell cycle progression was analysed by flow cytometry, DNA adduct formation was assessed by 32P-postlabeling analysis, microarrays of 44K human genome-wide oligos and RT-PCR were used to detect gene expression (mRNA) changes and Western blotting was performed to determine the expression of some proteins, including cytochrome P450 (CYP) 1A1 and CYP1B1, which are involved in BaP metabolism. RESULTS: Following BaP exposure, cells evaded G1 arrest and accumulated in S-phase. Higher levels of DNA damage occurred in S- and G2/M- compared with G0/G1-enriched cultures. Genes that were found to have altered expression included those involved in xenobiotic metabolism, apoptosis, cell cycle regulation and DNA repair. Gene ontology and pathway analysis showed the involvement of various signalling pathways in response to BaP exposure, such as the Catenin/Wnt pathway in G1, the ERK pathway in G1 and S, the Nrf2 pathway in S and G2/M and the Akt pathway in G2/M. An important finding was that higher levels of DNA damage in S- and G2/M-enriched cultures correlated with higher levels of CYP1A1 and CYP1B1 mRNA and proteins. Moreover, exposure of synchronised MCF-7 cells to BaP-7,8-diol-9,10-epoxide (BPDE), the ultimate carcinogenic metabolite of BaP, did not result in significant changes in DNA adduct levels at different phases of the cell cycle. CONCLUSIONS: This study characterised the complex gene response to BaP in MCF-7 cells and revealed a strong correlation between the varying efficiency of BaP metabolism and DNA damage in different phases of the cell cycle. Our results suggest that growth kinetics within a target-cell population may be important determinants of susceptibility and response to a genotoxic agent. Two-color Agilent array. A reference design was chosen that all samples were hybridised to universal human reference RNA (UHRR from stratagene). 8-condition experiment (4 conditions: G0/G1, S, G2/M and unsynchronised (N); 2 treatments: BaP and DMSO; 1 time point: 12 hours). Three biological replicates for each condition. One replicate per array.

背景：苯并[a]芘（Benzo[a]pyrene, BaP）是一种广泛分布的环境遗传毒性致癌物，可通过形成加合物损伤DNA。该类损伤联合芳香烃受体（aryl hydrocarbon receptor, AHR）的激活，可在细胞内诱导复杂的转录应答。为探究人类细胞在细胞周期的特定阶段是否对BaP更易感，研究人员将同步化的乳腺癌MCF-7细胞暴露于BaP环境中。本研究通过流式细胞术（flow cytometry）分析细胞周期进程，采用32P后标记法（32P-postlabeling analysis）评估DNA加合物的形成，使用44K全人类基因组寡核苷酸芯片（44K human genome-wide oligos）与逆转录聚合酶链反应（reverse transcription polymerase chain reaction, RT-PCR）检测基因表达（mRNA）变化，并通过蛋白质印迹法（Western blotting）测定部分蛋白质的表达水平，其中包括参与BaP代谢的细胞色素P450（cytochrome P450, CYP）1A1与CYP1B1。
结果：经BaP暴露后，细胞规避了G1期阻滞并在S期发生蓄积。与G0/G1富集培养物相比，S期和G2/M期培养物中的DNA损伤水平更高。表达发生改变的基因涉及异生物质代谢、细胞凋亡、细胞周期调控以及DNA修复等生物学过程。基因本体论（gene ontology, GO）与通路分析显示，多种信号通路参与了BaP暴露后的应答反应：包括G1期的连环蛋白/Wnt（Catenin/Wnt）通路、G1期与S期的细胞外调节蛋白激酶（extracellular regulated protein kinases, ERK）通路、S期和G2/M期的核因子红细胞2相关因子2（nuclear factor erythroid 2-related factor 2, Nrf2）通路，以及G2/M期的蛋白激酶B（Akt）通路。一项关键发现为，S期和G2/M期富集培养物中更高水平的DNA损伤，与CYP1A1和CYP1B1的mRNA及蛋白质表达水平呈显著正相关。此外，将同步化MCF-7细胞暴露于BaP的终末致癌代谢产物BaP-7,8-二醇-9,10-环氧化物（BPDE）后，不同细胞周期阶段的DNA加合物水平未出现显著变化。
结论：本研究阐明了MCF-7细胞对BaP的复杂基因应答，并揭示了BaP代谢效率的差异与不同细胞周期阶段的DNA损伤之间存在显著相关性。研究结果表明，靶细胞群体内的生长动力学可能是决定遗传毒性毒物易感程度与应答反应的重要因素。本实验采用双色安捷伦（Agilent）芯片，选用参考设计方案：所有样本均与通用人类参考RNA（universal human reference RNA, UHRR，购自Stratagene公司）进行杂交。实验包含8个条件：4种细胞周期状态（G0/G1、S、G2/M以及未同步化（N））、2种处理方式（BaP与二甲基亚砜（DMSO））、1个时间点（12小时）。每个条件设置3次生物学重复，每块芯片对应1次重复。