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.
| Milo, G E; D'Ambrosio, S; Kun, E (1989) Benzamide prevention of ultraviolet radiation-induced transformation as measured by anchorage-independent growth and the absence of correlation with thymidine dimer formation and DNA repair. Teratog Carcinog Mutagen 9:167-76 |
