Vandamme B, Liebaers I, Hens L, Bernheim J L, Roobol C
Laboratory for Experimental Chemotherapy, Faculty of Medicine and Pharmacy, Free University of Brussels, Belgium.
Hum Genet. 1988 Aug;79(4):341-6. doi: 10.1007/BF00282173.
The modes of action of 5-fluoro-2'-deoxyuridine (FdUrd) and 5-fluoro-2'-deoxycytidine (FdCyd) were studied in PHA-stimulated lymphocytes from normal volunteer donors and a fragile X patient. In both cell types, FdUrd and FdCyd inhibited cell proliferation at concentrations of 3 x 10(-8) M. Thymidylate synthetase was identified as the decisive target for the action of both FdUrd and FdCyd, as judged from the following observations: First, addition of thymidine to the culture medium was able to counteract both FdUrd and FdCyd toxicities, whereas addition of dCyd had no observable effect. Second, inhibition of the in situ thymidylate synthetase activity measured as an increase in the level of [3H]-dThd incorporation coincided with the inhibition of cell proliferation. Third, the inhibition of the thymidylate synthetase-dependent incorporation of [3H]-dUrd into newly synthesized DNA coincided with the inhibition of cell proliferation. The effects of FdUrd and FdCyd on the in vitro expression of fragile site Xq27 of fragile X chromosomes was shown to be based on the depletion of the intracellular pool of thymidine-5'-monophosphate (dTMP), as judged from the following observations: First, both the FdUrd- and FdCyd-dependent induction of site Xq27 coincided with the antiproliferative effects of the respective fluoropyrimidines. Second, addition of thymidine (dThd) to the culture medium both prevented the expression of site Xq27 and neutralized the cytotoxicity of FdUrd and of FdCyd. On the basis of these findings, we provide further evidence for the concept that the fragile X site is located in an AT-rich region.
研究了5-氟-2'-脱氧尿苷（FdUrd）和5-氟-2'-脱氧胞苷（FdCyd）对正常志愿者供体和一名脆性X综合征患者的PHA刺激淋巴细胞的作用方式。在这两种细胞类型中，FdUrd和FdCyd在3×10⁻⁸ M的浓度下抑制细胞增殖。从以下观察结果判断，胸苷酸合成酶被确定为FdUrd和FdCyd作用的决定性靶点：第一，向培养基中添加胸苷能够抵消FdUrd和FdCyd的毒性，而添加dCyd则没有可观察到的效果。第二，以[³H]-dThd掺入水平的增加来衡量的原位胸苷酸合成酶活性的抑制与细胞增殖的抑制相一致。第三，胸苷酸合成酶依赖性的[³H]-dUrd掺入新合成DNA的抑制与细胞增殖的抑制相一致。从以下观察结果判断，FdUrd和FdCyd对脆性X染色体脆性位点Xq27体外表达的影响是基于细胞内胸苷-5'-单磷酸（dTMP）池的耗竭：第一，FdUrd和FdCyd依赖性的位点Xq27诱导与各自氟嘧啶的抗增殖作用相一致。第二，向培养基中添加胸苷（dThd）既能阻止位点Xq27的表达，又能中和FdUrd和FdCyd的细胞毒性。基于这些发现，我们为脆性X位点位于富含AT区域这一概念提供了进一步的证据。
