The main objective of this research proposal is to use purified B and T lynphocytes from normal and leukemogenic humans as a model system to study critical molecular events that regulate excision (repair) and expression of chemically induced DNA adducts. Lymphocytes exist in a dynamic state that alternates between small quicescent cells and large antigen-activated proliferating cells. These cells require an activation step to become """"""""competent"""""""" to respond to a lineage specific growth factor receptor ligand which promotes cell cycle """"""""progression"""""""" and S phase entry. A battery of proximate carcinogens of diverse structure will be used to study whether 1) quiescent and proliferating normal B and T cells differ in their repair proficiency; 2) malignant human lymphocytes have defective repair pathways as compared to their normal counterparts; and 3) repair response of Go/G1 lymphocytes have any effects on normal proliferative response. The carcinogen DNA adducts and their repair will be quantitated by a previously described 32p post-labelling technique, which requires only 1-10 microgram of DNA and has recently been modified to detect on adduct in 10/9- 10/10 nucleotides. We will use freshly prepared peripheral blood lymphocytes, long-term cultured growth factor dependent cell lines and malignant primary and cultured cells, restricted at different stages of the cell cycle. In attempts to understand biological consequences of DNA damage, expression of c-myc, c- myb, and c-ras proto-oncogenes will be studies. A transient expression of these genes have been shown to be associated with the """"""""competent"""""""" and """"""""progression"""""""" phases of the cell cycle. In addition, studies will be initiated to clone human DNA repair genes using hybridization subtraction approaches of cDNA isolation. Therefore, forthcoming information from this grant proposal should provide relevant insights not only to the regulation of DNA repair, but also to the mechanism of carciogenesis in general.
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