The objective is to test the hypothesis that early S phase specificity for malignant transformation of cells by chemical carcinogens is due to the alteration of cellular oncogenes, at the time of their replication in the early S phase of the cell cycle. This hypothesis is supported by the 0ollowing observations: a) proliferating cells are very sensitive to chemical carcinogens and a higher frequency of transformation is observed when cells are treated at the beginning of the S phase; b) genes are replicated in S phase according to a specific temporal order that is maintained from one generation to the next; c) replicating DNA is more susceptible to chemical modifications by methylating carcinogens than bulk DNA; this characteristic, coupled to a smaller probability of repair before replication, should favor the fixation of genetic alterations; d) a recognized mechanism of activation of cellular oncogenes is through site-specific mutations. The above hypothesis will be tested by determining the time of replication of various cellular oncogenes, in several normal cells and their transformed counterparts, obtained in our laboratories according to defined protocols. The oncogenes to be studied in detail are the cellular homologs of v-erb, v-Ha-ras, v-Ki-ras, v-fos, v-sis and v-myc. To accomplish this task we will first develop a simple method for mapping the time of replication of specific genes. This method will be based on the enrichment of replicating sequences upon binding to nitrocellulose filters of DNA isolated from synchronized cells at different time points in the S phase. Subsequently, these filters will be blot-hybridized with specific gene-probes. The validity of this method will be checked by using DNA probes complementary to sequences that are known to be early- or late-replicating in fibroblasts. Furthermore, it will be used to verify whether transcription competence affects the time of replication of tissue-specific genes in fibroblasts, as compared to epithelial cells. We also consider important to compare the time of replication of oncogenes with their time of maximum transcription. It is conceivable that genes are also more susceptible to mutation by chemical carcinogens during a time when they are highly transcribed. Therefore, the results of these studies should help to elucidate the possible relationship between replication (or transcription) of cellular oncogenes and the higher frequency of transformation by chemical carcinogens in early S phase of the cell cycle.
|Doggett, N A; Cordeiro-Stone, M; Chae, C B et al. (1988) Timing of proto-oncogene replication: a possible determinant of early S phase sensitivity of C3H 10T1/2 cells to transformation by chemical carcinogens. Mol Carcinog 1:41-9|