The goal of this project is to better understand the extracellular and intracellular interactions between positive and negative regulators of hematopoietic stem cells (SC) as they pertain to the mechanism(s) of malignant transformation and to the development of therapeutically useful gen therapy protocols. To initiate and maintain the growth and differentiation of primitive progenitor cells, multiple cytokine stimulation is required. This has led to the concept of """"""""growth facto synergy"""""""". We show that such cooperativity also occurs between negative regulators of hematopoietic cell growth, and that the ability of primitive progenitors to proliferate depends on the balance of positive and negative signals the cell receives. Of several inhibitory cytokines studied only TGF-beta directly and reversibly inhibited all hematopoietic SC populations with marrow repopulating ability. TGF-B has inhibitory effects on the cell surface expression of many cytokine receptors that directly correlates with its effect on cell growth. SC factor receptor (c-kit) expression is down-regulated by TGF-beta in part by affecting c- kit mRNA stability. Also, TGF-beta prevents S phase cell cycle progression through an intracellular mechanism involving regulation of c- myb as well as c-myc. In vivo results demonstrated that TGF-beta can protect mice from both the lethal hematopoietic toxicity of 5-FU, as well as the non-hematopoiesis toxicity of DXR. The findings show that a negative regulator of hematopoiesis can be successfully used systemically to mediate chemoprotection in vivo. Several positive regulators of hematopoietic cells were used in vivo to determine their effect on myelopoiesis. The administration of rhIL-7 to mice twice a da for 7 days does not appreciably change BM cellularity, but does result in a 3-fold to 5-fold increase in the total number of leukocytes in the spleen and up to a 20-fold increase in the total number of peripheral WBC. Using our ability to regulate the growth of hematologic cells, we determined the feasibility of gene transfer under these conditions. We established that the ex-vivo transfer of the HS-tk suicide gene into T-cells before their infusion with hematopoietic SC could allow for selective in vivo depletion of these T-cells with ganciclovir (GCV) if the need subsequently arises.
