Abdel-Aziz W, Jiang H Y, Hickey R J, Malkas L H
University of Maryland School of Medicine, Department of Pharmacology and Experimental Therapeutics, Baltimore, MD 21201, USA.
Cancer Chemother Pharmacol. 2000;45(4):312-9. doi: 10.1007/s002800050046.
An intact and fully functional multiprotein DNA replication complex (DNA synthesome) from human as well as from murine mammary carcinoma cells was first isolated and characterized in our laboratory. The human cell synthesome supports the in vitro origin-specific simian virus 40 (SV40) DNA replication reaction in the presence of the viral large T-antigen using a semiconservative mechanism and has been shown to contain all the proteins and enzymes required to support DNA synthesis. We are currently using the DNA synthesome as a unique model for analyzing the mechanism of action of anticancer drugs affecting DNA replication. The purpose of this study was to further investigate the mechanism of action of ara-C using the DNA synthesome isolated from the human breast cancer cell line MDA MB-468.
Synthesome-mediated SV40 DNA replication was performed in the presence of various concentrations of ara-CTP (the active metabolite of ara-C) and the types of daughter DNA molecules produced were analyzed lusing neutral and alkaline gel electrophoresis. We also examined the effect of ara-C on intact MDA MB-468 cell DNA synthesis and on cell proliferation. In addition, we studied the effect of ara-CTP on the activity of some of the synthesome target proteins (the DNA polymerases alpha and delta).
Full-length daughter DNA molecules were obtained in the presence of low concentrations of ara-CTP while at higher concentrations, there was an inhibition of full-length daughter DNA synthesis. The findings suggest that specifically the initiation phase of DNA synthesis was inhibited by ara-CTP since the production of the short Okazaki fragments was suppressed at all concentrations of the drug above 10 microM. In addition, it was found that the IC50 of ara-CTP for inhibition of synthesome-mediated in vitro DNA replication was comparable to that required to inhibit intact cell DNA synthesis. Further experimentation has shown that ara-CTP preferentially inhibits the activity of the synthesome-associated DNA polymerase alpha enzyme while the DNA polymerase delta seems to be resistant to the inhibitory effect of that drug.
Our results indicate that ara-C's action on DNA replication is mediated primarily through DNA polymerase alpha and suggest that this enzyme plays a key role in DNA synthetic initiation events. The results also provide definitive support for the use of the DNA synthesome as a unique and powerful model for analyzing the mechanism of action of anticancer drugs which directly affect DNA replication.
人类以及小鼠乳腺癌细胞中完整且功能齐全的多蛋白DNA复制复合体（DNA合成体）最初是在我们实验室中分离并鉴定的。人类细胞合成体在病毒大T抗原存在的情况下，利用半保留机制支持体外起源特异性猿猴病毒40（SV40）DNA复制反应，并且已被证明包含支持DNA合成所需的所有蛋白质和酶。我们目前正在使用DNA合成体作为一种独特的模型来分析影响DNA复制的抗癌药物的作用机制。本研究的目的是使用从人乳腺癌细胞系MDA MB - 468中分离的DNA合成体，进一步研究阿糖胞苷（ara - C）的作用机制。
在存在各种浓度的阿糖胞苷三磷酸（ara - CTP，ara - C的活性代谢产物）的情况下进行合成体介导的SV40 DNA复制，并使用中性和碱性凝胶电泳分析产生的子代DNA分子类型。我们还研究了ara - C对完整的MDA MB - 468细胞DNA合成和细胞增殖的影响。此外，我们研究了ara - CTP对一些合成体靶蛋白（DNA聚合酶α和δ）活性的影响。
在低浓度ara - CTP存在下获得了全长子代DNA分子，而在较高浓度下，全长子代DNA合成受到抑制。这些发现表明，ara - CTP特异性抑制了DNA合成的起始阶段，因为在高于10微摩尔的所有药物浓度下，短冈崎片段的产生均受到抑制。此外，发现ara - CTP抑制合成体介导的体外DNA复制的IC50与抑制完整细胞DNA合成所需的IC50相当。进一步的实验表明，ara - CTP优先抑制与合成体相关的DNA聚合酶α酶的活性，而DNA聚合酶δ似乎对该药物的抑制作用具有抗性。
我们的结果表明，ara - C对DNA复制的作用主要通过DNA聚合酶α介导，并表明该酶在DNA合成起始事件中起关键作用。这些结果还为使用DNA合成体作为分析直接影响DNA复制的抗癌药物作用机制的独特而强大的模型提供了确凿支持。
