SITE SPECIFIC MODIFICATION OF HUMAN CELLULAR DNA

当人类细胞在细胞周期的早期S期接受致癌性损伤时， 细胞周期观察到对损伤的反应增强，如以下所证明的： 异常表型的表达增加。 然而，当一个 将无毒浓度的苯甲酰胺（BZ）加入细胞中， S期开始，随后用致癌物处理， 防止了异常表型。 致癌物-DNA加合物的测量 在BZ和非BZ处理的样品之间没有显示出任何差异。 研究 对B[a]P二醇环氧化物（BPDE-I）的结合的影响显示约. 3倍 BPDE-1与接头DNA的结合与BPDE-1的核心区相比， 染色质，在BZ的存在下。 与连接体的结合力相等 当BPDE-Ⅰ单独作用于S期细胞时，对细胞核DNA的表达有明显的抑制作用。 的 仅用BPDE-1处理的G细胞停滞中的融合细胞也结合了 致癌物优先于接头区。 这些数据表明，BZ 可以以某种方式保持DNA的接头和核心区域的完整性 在复制过程中，从而掩蔽DNA位点， 这是转型所必需的。 因为人类的转化能力 在S期早期加入致癌物， 苯甲酰胺的抑制作用在S期早期达到最大， 可以合理地假设BZ可以通过调节 致癌物质的特定DNA位点复制在此期间。 因此，我们的目标将试图审查BPDE I对以下方面的约束力： 处于细胞周期早期S期的细胞的复制和亲本DNA 以及BZ及其类似物对这种结合的影响。 我们将： 定量并比较复制DNA中特异性BPDE I-DNA加合物 和亲本DNA在早期S期;测量特异性BPDE I-DNA加合物 在复制叉相关的DNA在早期S期;测量 BPDE-1与复制DNA、亲本DNA的特异性结合， 复制叉相关的DNA时，细胞已被处理在早期S 在苯甲酰胺的存在下与致癌物相;研究结构 苯甲酰胺及其类似物对 它们对转化和BPDE-1与复制DNA结合的影响， 亲本DNA和复制叉相关DNA。 32 P后标记 技术首先由兰德拉斯开发，并由我们修改，以适应我们的 在这些研究中将使用实验来测量加合物。

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.