Both high and low dose rate x-irradiation of purified marrow stromal cells in vitro have been demonstrated by us to alter the growth of added purified hematopoietic stem cells. Dose rates of 5 or 20 rad/min (low) stabilize some parameters of hematopoiesis significantly better than an equivalent leukemia-cell lethal standardized dose delivered at 200 rad min (high), including: production and release from stromal cells of growth factors including granulocyte-macrophage colony stimulating factor-1 (FM-CSF-1) required for proliferation in agar overlay of committed granulocyte-macrophage progenitor cells (GM-CFUc). However, other parameters of hematopoiesis including cumulative production in liquid overlay of total hemopoietic cells and pluripotential hematopoietic stem cells (CFUs) are equally depressed at both low and high dose rates. These physiologic alterations of stromal cells are independent of proliferative integrity as measured by clonagenic survival of replated cells. The protective effect of low dose rate irradiation is lost by prior in vitro exposure of stromal cells to the chemotherapeutic alkylating agent, L-PAM. We will now determine the mechanism of these non-lethal physiologic effects of irradiation on marrow stromal cells, and the critical dose rate for each detectable biologic effect. Methods will involve continuous bone marrow cultures; preparation of purified stromal and hematopoietic stem cell cultures; identification of the phenotype of stromal cells surviving each irradiation dose; measurement of self-renewal capacity of CFUs; quantitation of levels of synthesis of multipotential cell CSF also known as Interleukin-3 (IL-3) and CSF-1; and study of the kinetics of repair of x-ray damage in these compared to other stromal cell cultures that have been treated prior to irradiation with the aklylating agent, L-Phenylalanine mustard; nitrosyl urea; adriamycin or busulfan in vitro. These studies should: improve understanding of irradiation effects that alter cell to cell interaction; and aid in modification of the sequence of chemotherapy and total body irradiation in drug/x-ray protocols to optimize clinical marrow engraftment and improve survival in bone marrow transplantion.
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