Ribovich M L, Kurian P, Milo G E
Carcinogenesis. 1986 May;7(5):737-44. doi: 10.1093/carcin/7.5.737.
Replicating DNA was modified by BPDE I to a greater extent than parental DNA when human fibroblast cells were treated with the carcinogen for 30 min in early S phase. Synchronized cells were exposed to 5-bromodeoxyuridine and treated with non-radioactive BPDE I and [methyl-3H]thymidine in early S phase. The density- and tritium-labeled, replicated DNA was separated from parental DNA in a CsCl gradient. The individual carcinogen-DNA adduct levels in both samples were quantitated by using the 32P-postlabeling method. The total modification of replicated DNA was 1.4-2.4 times greater than parental DNA. This difference was mainly reflected by differences in the main adducts, identified as the 3', [5'-32P]bisphosphates of 7R and 7S-BPDE I-dG. Confirmation of the identity of these two specific carcinogen-DNA adducts was accomplished by co-chromatography on t.l.c. with 3H-labeled 3',5'-bisphosphate adducts. The two 3H- and 32P-labeled adducts were isolated and dephosphorylated. The resultant 3H-labeled deoxyribonucleoside adducts were analyzed on h.p.l.c. and identified by co-chromatography with authentic standards. These results suggest that preferential modification of replicating DNA occurs when human cells are treated with BPDE I in early S phase. The ultimate result of this specific modification is the expression of a transformed phenotype.
当人类成纤维细胞在S期早期用致癌物处理30分钟时，复制中的DNA比亲代DNA受到BPDE I的修饰程度更大。同步化的细胞在S期早期暴露于5-溴脱氧尿苷，并分别用非放射性BPDE I和[甲基-³H]胸苷处理。在CsCl梯度中，将密度标记和氚标记的复制DNA与亲代DNA分离。使用³²P后标记法对两个样品中单个致癌物-DNA加合物的水平进行定量。复制DNA的总修饰程度比亲代DNA大1.4至2.4倍。这种差异主要体现在主要加合物的差异上，这些主要加合物被鉴定为7R和7S-BPDE I-dG的3'，[5'-³²P]双磷酸酯。通过与³H标记的3'，5'-双磷酸加合物在薄层层析上进行共色谱分析，确认了这两种特定致癌物-DNA加合物的身份。分离并去磷酸化了这两种³H和³²P标记的加合物。将所得的³H标记的脱氧核糖核苷加合物在高效液相色谱上进行分析，并通过与标准品共色谱进行鉴定。这些结果表明，当人类细胞在S期早期用BPDE I处理时，复制中的DNA会发生优先修饰。这种特异性修饰的最终结果是转化表型的表达。
