Binding of chlorozotocin and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea to chromatin and nucleosomal fractions of HeLa cells.
Binding of chlorozotocin and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea to chromatin and nucleosomal fractions of HeLa cells.
复制标题
氯脲佐菌素和 1-(2-氯乙基)-3-环己基-1-亚硝基脲与 HeLa 细胞染色质和核小体部分的结合。
作者：
K. Tew;S. Sudhakar;P. Schein;M. Smulson
Chlorozotocin和1-（2-chloroethyl）-3-cyclohexyl-1-nitrosourea（CCNU）都能特异性结合HeLa细胞基因组的延伸常染色质部分。对数期细胞与30 µm的每种放射性标记药物孵育2小时后，核染色质的烷基化（氯佐霉素和CCNU）和氨甲酰化（CCNU）均被限制在ECTHAM-纤维素色谱柱的后洗脱馏分中。这些数据通过用胰腺DNA酶I（优先切割转录活性染色质）酶消化药物处理的细胞核来证实。通过遵循消化动力学，烷基化染色质优先被酶溶解。此外，当与微球菌核酸酶digestine相比时，氯脲霉素和CCNU都显示出与核小体核心颗粒相关的DNA烷基化。
在定量上，氯脲霉素的染色质烷基化作用是环己亚硝脲的两倍。用5.0 mm丁酸钠预处理对数期细胞，通过引起组蛋白修饰和转录活性增加来影响染色质结构。同时，两种亚硝基脲的细胞摄入量增加了两倍。此外，染色质烷基化和组蛋白和非组蛋白蛋白的氨甲酰化增加了2 - 3倍。
在浓度为30 µm CCNU时，核蛋白的氨甲酰化仅限于非组蛋白组分。只有当染色质悬浮液（用微球菌核酸酶温和消化细胞核制备）用0.3 mm CCNU处理时，才能检测到组蛋白的烷基化。
Both chlorozotocin and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) bind specifically to the extended euchromatin fraction of the HeLa cell genome. After a 2-hr incubation of log-phase cells with each radiolabeled drug at 30 µm, both alkylation (chlorozotocin and CCNU) and carbamoylation (CCNU) of nuclear chromatin were shown to be confined to late-eluting fractions from an ECTHAM-cellulose chromatography column. These data were confirmed by enzymatic digestion of drug-treated nuclei with pancreatic DNase I (preferentially cleaves transcriptionally active chromatin). By following the kinetics of digestion, the alkylated chromatin was preferentially solubilized by the enzyme. In addition, when compared to micrococcal nuclease digestions, both chlorozotocin and CCNU were shown to alkylate the DNA associated with the nucleosome core particle.
Quantitatively, chromatin alkylation by chlorozotocin was twice that of CCNU. Pretreatment of log-phase cells with 5.0 mm sodium butyrate affected the chromatin structure by causing histone modifications and an increase in transcriptional activity. Concomitantly, the cellular uptake of both nitrosoureas was increased twofold. Also, the chromatin alkylation and histone and nonhistone protein carbamoylation were increased two- to threefold.
At a concentration of 30 µm CCNU, carbamoylation of nuclear proteins was restricted to the nonhistone fractions. Alkylation of histone proteins was detected only when a chromatin suspension (prepared by mild digestion of nuclei with micrococcal nuclease) was treated with 0.3 mm CCNU.