Attenuation of drug-stimulated topoisomerase II DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIα
Attenuation of drug-stimulated topoisomerase II DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIα
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DOI：
10.1042/bj3360727
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发表时间：
1998-12-15
影响因子：
4.1
通讯作者：
Ganapathi, R
中科院分区：
文献类型：
--
作者：
Aoyama, M;Grabowski, DR;Ganapathi, R
拓扑异构酶 II (topo II) 是细胞活力的必需酶，也是临床上重要的抗肿瘤药物的靶标，可刺激 toro II 介导的 DNA 断裂。研究了 toro II α 磷酸化改变的作用及其对药物诱导的 DNA 损伤和细胞毒性的影响。用钙缓冲液 1,2-双-(邻氨基苯氧基)乙烷-N,N,N',N'-四乙酸四(乙酰氧基甲基)酯 (BAPTA-AM) 加载 HL-60 细胞后，可消除细胞内 Ca2+ 瞬变，依托泊苷 (VP-16) 或安吖啶 (m-AMSA) 稳定的拓扑结构显着减少，II-DNA 可裂解观察到复合物形成和细胞毒性相应降低。在无细胞系统中，当检测 VP-16 稳定的拓扑 II-DNA 复合物形成时，BAPTA-AM 处理细胞的核提取物表现出明显较低的活性，但未检测动质体 DNA 的去连接。相比之下，在 HL-60 细胞中负载 N,N,N',N'-四-(2-吡啶基)乙二胺 (TPEN)（可结合重金属而不干扰钙或镁浓度）不会显着影响 VP-16 刺激的拓扑 II-DNA 可裂解复合物形成或细胞毒性。在 HL-60 细胞中，BAPTA（而非 TPEN）的积累也导致了 toro II α 的低磷酸化。 HL-60 细胞拓扑 IIa 蛋白的胰蛋白酶磷酸化肽图谱显示：(a) 未处理细胞中的 8 个主要磷酸化位点； (b) BAPTA-AM处理的细胞中八个位点中的两个位点低磷酸化； (c) 抗拓扑 II 毒 HL-60 细胞中八个位点中的 2 到 4 个位点发生低磷酸化。 BAPTA-AM 处理野生型细胞后出现的两个低磷酸化位点与抗性细胞中的低磷酸化位点相同，但与酪蛋白激酶 II 的底物位点不同 [Wells, Addison, Fry, Ganapathi 和 Hickson (1994) J. Biol.化学，269, 29746-29751]。总之，细胞内 Ca2+ 瞬变的变化导致拓扑 II α 位点特异性低磷酸化，可能参与调节拓扑 II 毒物引起的 DNA 损伤和细胞毒性潜力。
Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate toro II-mediated DNA scission. The role of alterations in toro II alpha phosphorylation and its effect on drug-induced DNA damage and cytotoxicity were investigated. Follow ing loading of HL-60 cells with the calcium buffer 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetra(acetoxymethyl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a significant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized topo, II-DNA cleavable complex formation and a corresponding decrease in cytotoxicity was observed, In a cell-free system, nuclear extracts from BAPTA-AM-treated cells exhibited markedly less activity when assayed for VP-16-stabilized topo II-DNA complex formation, but not decatenation of kinetoplast DNA. In contrast, the loading of HL-60 cells with N,N,N',N'-tetrakis-(2-pyridyl)ethylenediamine (TPEN), which binds heavy metals without disturbing calcium or magnesium concentrations, did not significantly affect VP-16-stimulated topo II-DNA cleavable complex formation or cytotoxicity. In HL-60 cells the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylation of toro II alpha. Tryptic phosphopeptide mapping of topo IIa protein from HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treated cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated sites present following BAPTA-AM treatment of wild-type cells were identical with the hypophosphorylated sites in the resistant cells, but were not the same as the sites that are substrates for casein kinase II [Wells, Addison, Fry, Ganapathi and Hickson (1994) J. Biol. Chem, 269, 29746-29751]. In summary, changes in intracellular Ca2+ transients that lead to the site-specific hypophosphorylation of topo II alpha are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons.