Inhibition of DNA replication by tirapazamine.
Inhibition of DNA replication by tirapazamine.
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发表时间：
2001-07
期刊：
影响因子：
11.2
通讯作者：
Katherine B. Peters;Hongyan Wang;J. Brown;G. Iliakis
中科院分区：
文献类型：
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作者：
Katherine B. Peters;Hongyan Wang;J. Brown;G. Iliakis
替拉扎明 (TPZ) 是一种缺氧选择性细胞毒素，目前正在与放疗和基于顺铂的化疗相结合进行 II 期和 III 期临床试验。还原酶将 TPZ 转化为细胞毒性自由基，在缺氧条件下产生 DNA 损伤。因为负责 TPZ 生物活化的一种或多种酶被认为位于核基质处或附近，因此我们假设 TPZ 可能对 DNA 复制产生重大影响，而 DNA 复制是已知主要发生在核基质处的过程。为了评估 TPZ 对 DNA 复制的影响，我们测量了放射性胸苷掺入 HCT116 人类结肠癌细胞和 HeLa 细胞 DNA 中的情况。我们发现，在缺氧条件下用 TPZ 预处理的细胞中，放射性胸苷的掺入受到显着抑制。 TPZ 诱导的 DNA 合成抑制作用比有毒剂量的电离辐射产生的抑制作用要大得多。我们使用基于 SV40 的体外 DNA 复制测定来研究 TPZ 处理的细胞中 DNA 合成的抑制机制。通过该测定，我们发现在缺氧条件下用 TPZ 处理的细胞制备的提取物的 DNA 复制活性仅为对照细胞中测量的 25-50%。这种 DNA 复制活性的降低与用于体外 DNA 复制测定的细胞质提取物中复制蛋白 A (RPA) 水平的降低有关，并且可以通过添加重组人 RPA 来克服。此外，我们通过间接免疫荧光表明，TPZ 导致 RPA (RPA2) 的 p34 亚基定位于小的亚核灶。这些结果表明，TPZ 显着抑制 DNA 复制，并且抑制机制至少部分涉及 RPA 的变化，从而改变其细胞定位。
Tirapazamine (TPZ) is a hypoxia-selective cytotoxin that is currently being examined in Phase II and III clinical trials in combination with radiotherapy and cisplatin-based chemotherapy. Reductases convert TPZ to a cytotoxic radical that produces DNA damage under hypoxic conditions. Because one or more of the enzymes responsible for the bioactivation of TPZ is/are thought to be at or near the nuclear matrix, we hypothesized that TPZ may have a major affect on DNA replication, a process that is known to occur predominantly at the nuclear matrix. To assess the effect of TPZ on DNA replication, we measured the incorporation of radioactive thymidine into DNA of HCT116 human colon cancer cells and HeLa cells. We show that incorporation of radioactive thymidine is dramatically inhibited in cells that are pretreated with TPZ under hypoxic conditions. TPZ-induced inhibition of DNA synthesis was much greater than that produced by more toxic doses of ionizing radiation. We used the SV40-based in vitro DNA replication assay to study the mechanism of inhibition of DNA synthesis in cells treated with TPZ. Using this assay, we show that extracts prepared from cells treated with TPZ under hypoxic conditions had only 25-50% of the DNA replication activity measured in control cells. This reduction in DNA replication activity was associated with a reduction in levels of replication protein A (RPA) in cytoplasmic extracts used for the in vitro DNA replication assay and could be overcome by addition of recombinant human RPA. Furthermore, we show by indirect immunofluorescence that TPZ leads to a localization of the p34 subunit of RPA (RPA2) to small subnuclear foci. These results show that TPZ dramatically inhibits DNA replication and that the mechanism of inhibition, at least in part, involves changes in RPA that alter its cellular localization.