SITE SPECIFIC MODIFICATION OF HUMAN CELLULAR DNA

当人类细胞在S早期受到致癌侮辱时， 细胞周期对侮辱的高度反应被观察到，证据是 异常表型的表达增加。然而，当一个 在细胞中加入无毒浓度的苯甲酰胺(BZ) 起病的S期后用致癌物治疗，其表达 防止了异常表型。致癌物-DNA加合物的测定 不显示BZ处理的样品和非BZ处理的样品之间的任何差异。研究 关于B[a]P二醇环氧化物(BPDE-I)的结合，揭示了约3倍 BPDE-I与接头DNA的结合与核心区的比较 染色质，在BZ存在下。与连接子有同样的结合。 S期细胞单独用BPDE-I处理时的核心DNA。这个 仅用BPDE-I处理的G细胞停滞中的融合细胞也结合了 致癌物优先于连接区。这些数据表明，BZ 可能会以某种方式保持DNA连接子和核心区的完整性 在复制期间，从而掩蔽DNA位点，修改 这是变形所必需的。因为它的转化能力 S期早期加入致癌物，致癌物增多 而在S前期，苯甲酰胺的抑制作用最大，它 合理地假设BZ可以通过调节 致癌物与在此期间复制的特定DNA位点有关。 因此，我们的目标将试图检查BPDE I对 细胞周期早期S期细胞的复制和亲本DNA 以及BZ及其类似物对这种结合的影响。我们会： DNA复制中特异的BPDE I-DNA加合物的定量和比较 和亲本DNA在S早期；测定特异的BPDE I-DNA加合物 在S早期复制叉状相关的DNA中，测量 BPDE-I与复制DNA、亲本DNA和 S早期细胞处理后复制叉状DNA的研究 在苯甲酰胺存在下与致癌物的相；研究其结构 苯甲酰胺及其类似物对苯丙氨酸的活性关系 它们对转化和BPDE-I与复制DNA的结合的影响， 父母的DNA和复制的叉子相关DNA。32P-后标记 技术最先由Randerath开发，并由我们进行修改以适应我们的 实验将被用来测量这些研究中的加合物。

When human cells receive a carcinogenic insult in the early S phase of the cell cycle a heightened response to the insult is observed as evidenced by the increased expression of an abnormal phenotype. However, when a non-toxic concentration of benzamide (BZ) is added to the cells at the onset of S phase followed by treatment with a carcinogen, the expression of abnormal phenotype is prevented. Measurement of carcinogen-DNA adducts did not show any difference between the BZ and non-BZ treated samples. Studies on the binding of B[a]P diol epoxide (BPDE-I) revealed ca. 3 times more binding of BPDE-I to the linker DNA compared to the core region of the chromatin, in the presence of BZ. There was equal binding to the linker and core DNA when the cells in S phase were treated with BPDE-I alone. The confluent cells in G cell arrest treated only with BPDE-I also bound the carcinogen preferentially to the linker region. These data suggest that BZ may somehow retain the integrity of the linker and core region of the DNA during replication, thereby masking the DNA sites, the modification of which is necessary for tranformation. Since the transforming ability of the carcinogen is increased when the carcinogen is added in early S phase and the inhibitory effect by benzamide is maximum during early S phase, it is reasonable to assume that BZ may act by regulating the binding of the carcinogen to the specific DNA sites replicated during this period. Therefore, our objectives will attempt to examine the binding of BPDE I to replicating and parental DNA of cells in early S phase of the cell cycle and the effect of BZ and its analogues on this binding. We will: quantitate and compare the specific BPDE I-DNA adducts in replicating DNA and parental DNA in early S phase; measure the specific BPDE I-DNA adducts in replicating fork-associated DNA during early S phase; measure the specific binding of BPDE-I to the replicating DNA, parental DNA, and replicating fork-associated DNA when the cells have been treated in early S phase with the carcinogen in the presence of benzamide; study the structure activity relationship between benzamide and its analogues with regard to their effects on transformation and BPDE-I binding to replicating DNA, parental DNA, and replicating fork-associated DNA. The 32P-postlabeling technique first developed by Randerath and modified by us to suit our experiments will be used to measure the adducts in these studies.