The goal of this project is to better understand the extracellular signals and intracellular interactions between positive and negative regulators of hematopoietic stem cells as they pertain to the mechanism(s) of malignant transformation and to the development of therapeutically useful gene therapy protocols. We and others have shown that initiate and maintain the growth and differentiation of primitive progenitor cells, multiple cytokine stimulation (growth factor synergy) is required. Recently, we showed that such cooperativity also occurs between negative regulators of cell growth, and that the ability of primitive progenitors to proliferate depends on the balance of positive and negative signals the cell receives. Transforming growth factor-b (TGF-b) directly and reversibly inhibits hematopoietic stem cells with marrow repopulating ability. Also, short-term incubation with TGF-b does not damage the self-renewal potential of these stem cells. TGF-a has inhibitory effects on the cell surface expression of many cytokine receptors that directly correlates with its effect on cell growth. For example, stem cell factor receptor (c-kit) expression is down-regulated by TGF-b in part by affecting c-kit mRNA stability. Also, TGF-b prevents S phase cell cycle progression through an intracellular mechanism involving regulation of transcription factors and cell cycle regulatory proteins. In vivo results demonstrated that TGF-b can protect mice from both the lethal hematopoietic toxicity of 5-FU, as well as the non- hematopoiesis toxicity of DXR. These findings show that a negative regulator of hematopoiesis can be successfully used systemically to mediate chemoprotection in vivo. Also, positive regulators of hematopoiesis were used in vivo to determine their effect on myelopoiesis. Administration of recombinant human interleukin-7 (rhIL-7) to mice resulted in up to a 20-fold increase in the total number of peripheral white blood cells (PBL). These PBL contained increased number of stem cells with lomg-term marrow repoulating ability.Using our ability to regulate the growth of hematologic cells, the feasibility of gene transfer was studied under these conditions. We showed that the ex- vivo transfer of the herpes simplex thymidine kinase suicide gene into T cells before their infusion with hematopoietic stem cells could allow for selective in vivo depletion of these T cells with ganciclovir. Thus, cytokine regulation of stem cells might be therapeutically useful.
