Prolonged Inhibition by X-Rays of DNA Synthesis in Cells Obtained by Transformation of Primary Rat Embryo Fibroblasts with Oncogenes H-ras and v-myc

Abstract

Transfection of primary rat embryo fibroblasts with the H-ras oncogene plus the cooperating oncogene v-myc results in the development of foci of morphologically altered tumorigenic cells. We examined radiation (X-rays) induced inhibition of DNA synthesis in cell lines derived from such transformed clones and compared the results to those obtained with the nontransformed parental cells, rat embryo fibroblasts, as well as with cells immortalized either spontaneously, or after transfection with nuclear oncogenes (v-myc, E1A). Inhibition by X-rays of DNA synthesis was higher and persisted for longer periods of time in the H-ras- plus v-myc-transformed cell lines as compared to their nontransformed counterparts. When the rate of DNA synthesis was measured as a function of dose 3 h after irradiation, biphasic curves were observed in all cell lines tested with a radiation sensitive and a radiation resistant component, known to correspond to inhibition of replicon initiation and chain elongation, respectively. A substantially larger inhibition of DNA synthesis was observed between 0 and 30 Gy in H-ras- plus v-myc-transformed cell lines, as compared to their nontransformed counterparts, presumably caused by sustained inhibition of replicon initiation. Hypersensitive DNA synthesis to X-rays was also observed in a transformed cell line obtained by transfection of rat embryo fibroblasts with H-ras in cooperation with the oncogene E1A, but normosensitive DNA synthesis in a rare transformant obtained by transfection with H-ras alone. These results suggest a direct or indirect involvement of the oncogene H-ras in cooperation with the oncogene v-myc (or other nuclear oncogenes such as E1A) in the control of DNA synthesis in irradiated cells. This control of DNA synthesis may be mediated via a trans-acting mechanism that involves the production of a diffusible factor in response to the radiation insult, or, by a cis-acting mechanism that directly affects the replication machinery. Circumstantial evidence for possible involvement of oncogenes of the ras and myc families in DNA synthesis support this hypothesis. There was an inverse correlation between sensitivity to radiation-induced killing and prolonged inhibition by radiation of DNA synthesis, with radioresistant cell lines displaying longer inhibition of DNA synthesis. However, inhibition by radiation of DNA synthesis was similar in normal human fibroblasts (W138) and cells derived from a radiation-resistant human carcinoma cell line (SQ-20B) suspected to carry an abnormal c-raf-1 oncogene. In addition, inhibition of DNA synthesis in SQ20B cells was similar to that of SCC61 cells, a radiation-sensitive cell line derived from a carcinoma of the tongue.