Meechan P J, Vaughan A T, Giometti C S, Grdina D J
Department of Biological Sciences, Northern Illinois University, DeKalb 60115.
Radiat Res. 1991 Feb;125(2):152-7.
The radioprotector WR-1065 (N-(2-mercaptoethyl)-1,3-diaminopropane) has been shown to be the active moiety involved in protecting mammalian cells from the cytotoxic and mutagenic effects of ionizing radiation after administration of WR-1065 or the phosphorylated form, WR-2721. Initial experiments demonstrated that, in our hands, WR-1065 protects Chinese hamster AA8 cells from killing by (a) mechanism(s) other than induction of hypoxia. AA8 cells were then incubated in the presence of [14C]WR-1065 to determine whether association of WR-1065 in vivo was random or targeted to the nucleus or the nuclear matrix. The kinetics of incorporation of labeled material showed rapid incorporation for the first 30 min and little, if any, additional incorporation over the next 2.5 h. Examination of nuclei and nucleoids generated from the AA8 cells indicated that approximately 10% of the drug was localized in the nucleus and the drug that remained was not dislodged with repeated washes of the filters. Association kinetics of the drug with nuclei and nucleoids indicated that there was little increase in drug association with time, suggesting that there may be a limited number of strong association sites in the nucleus, but these sites are either with DNA or with matrix proteins. Exposure of the AA8 cells to 6 Gy of 60Co gamma rays did not significantly alter the association of the drug with AA8 cells. Incubating AA8 cells in [14C]WR-1065 for 30 min and then incubating in drug-free medium indicated that nearly all of the drug was lost from cells within the first 5 min of incubation in drug-free medium. The low level of tightly bound matrix-associated label may be important in generating alterations in matrix organization that have been observed previously in this laboratory.
辐射防护剂WR-1065（N-(2-巯基乙基)-1,3-二氨基丙烷）已被证明是在给予WR-1065或其磷酸化形式WR-2721后，参与保护哺乳动物细胞免受电离辐射的细胞毒性和诱变作用的活性部分。初步实验表明，在我们的实验中，WR-1065通过一种或多种非诱导缺氧的机制保护中国仓鼠AA8细胞免受杀伤。然后将AA8细胞在[14C]WR-1065存在下孵育，以确定WR-1065在体内的结合是随机的还是靶向细胞核或核基质。标记物掺入的动力学表明，在最初的30分钟内掺入迅速，在接下来的2.5小时内几乎没有额外的掺入（如果有也是极少的）。对AA8细胞产生的细胞核和核小体的检查表明，约10%的药物定位于细胞核，剩余的药物不会因滤膜的反复洗涤而被去除。药物与细胞核和核小体的结合动力学表明，药物结合随时间几乎没有增加，这表明细胞核中可能存在有限数量的强结合位点，但这些位点要么与DNA结合，要么与基质蛋白结合。将AA8细胞暴露于6 Gy的60Coγ射线不会显著改变药物与AA8细胞的结合。将AA8细胞在[14C]WR-1065中孵育30分钟，然后在无药物培养基中孵育，结果表明，在无药物培养基中孵育的最初5分钟内，几乎所有的药物都从细胞中丢失了。与基质紧密结合的低水平标记物可能在产生本实验室先前观察到的基质组织改变方面很重要。
