The repair of radiation-induces DNA damage in mammalian cells has usually been investigated by using proliferating cells grown in vitro. The vast majority of mammalian cells in situ, however, are post-mitotic existing in a state of terminal differentiation. The general aim of the proposed research is to investigate the repair of radiation-induced DNA damage on the molecular, biochemical and functional levels in terminally differentiated cells. As a model for cellular differentiation, these studies will focus on the murine 3T3-T proadipocyte cell system. In this in vitro system, exposure of actively cycling 3T3-T proadipocytes to human plasma results in their terminal differentiation into mature adipocytes. Preliminary studies indicate that the differentiation of 3T3-T stem cells into adipocytes is accompanied by a reduced capacity to repair DNA damage induced by radiation, both ionizing and UV.
One aim of this proposal is to define the mechanism(s) responsible for the reduced repair capabilities of terminally differentiated 3T3-T cells. These studies will consist of evaluating the repair of the various types of radiation-induced DNA lesions and investigating various parameters that may influence their repair. In addition to these mechanistic studies, a primary goal of this proposal is to investigate the biological consequence of the DNA repair deficiency in terminally differentiated 3T3-T cells. Because transcription is required to maintain the function of differentiated cells, RNA synthesis, specific enzyme activities and mRNA levels will be evaluated after irradiation. DNA degradation will also be used as a measure of the functional integrity of a differentiated cell.
The final aim of this proposal involves determining the effects of differentiation on the repair of specific genomic sequences. The repair of UV-induced pyrimidine dimers will be analyzed in genes that are turned on and off during 3T3-T cell differentiation. Investigators of this nature are designed to determine the influence of gene expression on DNA repair. Thus, these studies should not only generate information regarding the influence of unrepaired DNA damage on the function of post- mitotic, differentiated cells, but also contribute to the basic understanding of the regulation of DNA repair processes. In addition, these investigations should provide data on which to base future hypotheses regarding the potential relationship between DNA damage in endstage, functional cells and radiotherapy-induced normal tissue damage.
