ONCOGENES AND THE RADIOSENSITIVITY OF DNA REPLICATION

点击翻译按钮获取中文摘要

The present proposal is based on the novel observation that radiation exposure inhibits DNA replication more persistently and to a larger extent in primary rat embryo fibroblasts (REF) transfected with the oncogenes H-ras plus v-myc, as compared to REF or, to cell lines obtained from REF by transfection with either oncogene alone. It is designed to study the involvement of oncogenes of the ras and myc families and of protein phosphorylation on these late events f regulation of DNA replication in irradiated cells. The model we test is based on the hypothesis that DNA damage initiates a transduction pathway that transmits signals from myc to ras which in turn activates unknown protein kinases that reduce p34cdc2 kinase activity, and as a result initiation of DNA replication. It assumes that this regulation utilizes components identified as parts of the regulatory pathway for initiation of DNA replication in non-irradiated cells, and speculates that the function of these regulatory components is affected by activation and/or overexpression of proto-oncogenes. There is evidence suggesting cooperation between ras and myc in the initiation of transduction pathways, and ras is known to activate different families of protein kinases. Furthermore, recent evidence points to p34cdc2 kinase as a key regulator in the initiation events of DNA replication. The proposed research is expected to provide information on the contribution of oncogene activation to the genetic control of DNA replication. It provides the first steps towards characterization of important response pathways in irradiated cells and examines the role of p34cdc2 kinase activity in the process. The work involves the study of the effect of oncogenes of the families ras and myc alone or in combination, on the regulation of DNA replication in primary REF and immortalized NIH 3T3 cells. Wild type and mutant forms of these oncogenes will be used. Constitutive and conditional vectors will be employed as tools to test the effect of expression level on radiation induced inhibition of DNA replication. The correlation will be further tested by the use of vectors expressing antisense RNA and ribozymes directed against the transcripts of these oncogenes. The contribution of protein kinases in the proposed mechanism will be tested by examining the modulations induced in DNA replication after incubation of irradiated cells with compounds that either increase or decrease their activity. Finally, two model systems for DNA replication utilizing cell extracts and plasmids carrying the simian virus SV40 origin sequences, or isolated nuclei will be used in a attempt to identify proximate effects of DNA replication ultimately affected by the expression of oncogenes. A wealth of preliminary experimental evidence is provided supporting our earlier observations and the hypotheses on which this proposal rests. The existence e of factors that control DNA replication in irradiated cells has been suggested by experiments with cells from ataxia telangiectasia (AT) patients. Probably activation of such pathways allows the cell to successfully respond to environmental insults that damage its DNA, and to reduce their adverse consequences. Deregulation of DNA replication is a fundamental alteration occurring in a normal cell in the process of its conversion to a malignant cell.