Laboratory of Molecular Basis of Ageing, Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warszawa, Poland.
Faculty of Chemistry, Rzeszow University of Technology, 6 Powstancow Warszawy Ave., 35-959 Rzeszow, Poland.
Mech Ageing Dev. 2018 Mar;170:22-29. doi: 10.1016/j.mad.2017.07.006. Epub 2017 Jul 21.
The response of human colon cancer C85 cells to methotrexate takes the form of reversible growth arrest of the type of stress-induced senescence. In the present study it is shown that during C85 cell progression into methotrexate-induced senescence, dihydrofolate reductase, the primary intracellular target for the drug, is stabilized at the protein level and its enzymatic activity, assayed in crude cellular extracts, decreases by 2-fold. Dihydrofolate reductase inhibition results in an increase in dihydrobiopterin level and an ultimate decrease in the tetrahydrobiopterin: dihydrobiopterin ratio in senescent cells. Endothelial nitric oxide synthase expression declines. Despite concomitant upregulation of inducible nitric oxide synthase expression, no nitric oxide generation in senescent cells is detected. Progressing oxidative stress accompanies establishment of the state of senescence. DNA damage, in the form of double strand-breaks, occurs at the highest level at the senescence initiation phase and decreases as cells progress into the senescence maintenance phase.
人结肠癌 C85 细胞对甲氨蝶呤的反应表现为应激诱导衰老的可逆生长停滞。本研究表明，在 C85 细胞向甲氨蝶呤诱导的衰老进展过程中，二氢叶酸还原酶（药物的主要细胞内靶标）在蛋白质水平上稳定，其在粗细胞提取物中测定的酶活性降低了 2 倍。二氢叶酸还原酶抑制导致二氢生物蝶呤水平增加，并最终降低衰老细胞中的四氢生物蝶呤：二氢生物蝶呤比值。内皮型一氧化氮合酶表达下降。尽管诱导型一氧化氮合酶表达同时上调，但在衰老细胞中未检测到一氧化氮生成。随着衰老状态的建立，进行性氧化应激随之发生。以双链断裂形式存在的 DNA 损伤在衰老起始阶段达到最高水平，并随着细胞进入衰老维持阶段而降低。
