Bullinger Dino, Neubauer Hans, Fehm Tanja, Laufer Stefan, Gleiter Christoph H, Kammerer Bernd
Department of Pharmacology and Toxicology, Division of Clinical Pharmacology, University Hospital Tübingen, Otfried-Müller-Str, 45, 72076 Tübingen, Germany.
BMC Biochem. 2007 Nov 29;8:25. doi: 10.1186/1471-2091-8-25.
Cancer, like other diseases accompanied by strong metabolic disorders, shows characteristic effects on cell turnover rate, activity of modifying enzymes and DNA/RNA modifications, resulting also in elevated amounts of excreted modified nucleosides. For a better understanding of the impaired RNA metabolism in breast cancer cells, we screened these metabolites in the cell culture supernatants of the breast cancer cell line MCF-7 and compared it to the human mammary epithelial cells MCF-10A. The nucleosides were isolated and analyzed via 2D-chromatographic techniques: In the first dimension by cis-diol specific boronate affinity extraction and subsequently by reversed phase chromatography coupled to an ion trap mass spectrometer.
Besides the determination of ribonucleosides, additional compounds with cis-diol structure, deriving from cross-linked biochemical pathways, like purine-, histidine- and polyamine metabolism were detected. In total, 36 metabolites were identified by comparison of fragmentation patterns and retention time. Relation to the internal standard isoguanosine yielded normalized area ratios for each identified compound and enabled a semi-quantitative metabolic signature of both analyzed cell lines.13 of the identified 26 modified ribonucleosides were elevated in the cell culture supernatants of MCF-7 cells, with 5-methyluridine, N2,N2,7-trimethylguanosine, N6-methyl-N6-threonylcarbamoyladenosine and 3-(3-aminocarboxypropyl)-uridine showing the most significant differences. 1-ribosylimidazole-4-acetic acid, a histamine metabolite, was solely found in the supernatants of MCF-10A cells, whereas 1-ribosyl-4-carboxamido-5-aminoimidazole and S-adenosylmethionine occurred only in supernatants of MCF-7 cells.
The obtained results are discussed against the background of pathological changes in cell metabolism, resulting in new perspectives for modified nucleosides and related metabolites as possible biomedical markers for breast carcinoma in vivo.
癌症与其他伴有强烈代谢紊乱的疾病一样，对细胞更新率、修饰酶活性及DNA/RNA修饰具有特征性影响，这也导致排泄的修饰核苷量增加。为了更好地理解乳腺癌细胞中受损的RNA代谢，我们在乳腺癌细胞系MCF - 7的细胞培养上清液中筛选了这些代谢物，并将其与人类乳腺上皮细胞MCF - 10A进行比较。通过二维色谱技术分离并分析核苷：在第一维中通过顺二醇特异性硼酸酯亲和萃取，随后通过与离子阱质谱仪联用的反相色谱法。
除了测定核糖核苷外，还检测到了其他具有顺二醇结构的化合物，这些化合物源自交联的生化途径，如嘌呤、组氨酸和多胺代谢。通过比较碎片模式和保留时间，总共鉴定出36种代谢物。与内标异鸟苷的关系得出了每种鉴定化合物的归一化面积比，并实现了两种分析细胞系的半定量代谢特征。在鉴定出的26种修饰核糖核苷中，有13种在MCF - 7细胞的细胞培养上清液中升高，其中5 - 甲基尿苷、N2,N2,7 - 三甲基鸟苷、N6 - 甲基 - N6 - 苏氨甲酰腺苷和3 - (3 - 氨基羧丙基) - 尿苷表现出最显著的差异。组胺代谢物1 - 核糖基咪唑 - 4 - 乙酸仅在MCF - 10A细胞的上清液中发现，而1 - 核糖基 - 4 - 羧酰胺 - 5 -氨基咪唑和S - 腺苷甲硫氨酸仅在MCF - 7细胞的上清液中出现。
根据细胞代谢的病理变化背景对所得结果进行了讨论，这为修饰核苷及相关代谢物作为乳腺癌体内可能的生物医学标志物带来了新的视角。
