Role of bone marrow stromal cells in irradiation leukemogenesis.

The role of bone marrow stromal cells of the hematopoietic microenvironment in ionizing-irradiation leukemogenesis is a focus of current investigation. Evidence from recent in vitro and in vivo experiments suggests that damage by slowly proliferating cells of the hematopoietic microenvironment contributes to the sustained survival of irradiation-damaged hematopoietic progenitor cells/stem cells and can contribute to the selection and proliferation of a malignant clone. The molecular mechanism of the interaction of irradiated stromal cells with attached hematopoietic cells has been difficult to evaluate. Irradiated bone marrow stromal cell line D2XRII demonstrated altered patterns of fibronectin splicing and increased expression of several transcriptional splice variants of macrophage-colony-stimulating factor. Differential display has revealed specific radiation-induced gene transcripts which persist after irradiation of stromal cells in vitro or in vivo. In recent experiments, we demonstrated that irradiation of mouse bone marrow stromal cell line D2XRII induces release of significant levels of transforming growth factor (TGF)-beta into the tissue culture medium despite the lack of a detectable increase in TGF-beta mRNA. Since TGF-beta is known to induce reactive oxygen species (ROS), we tested how a target hematopoietic cell line, responsive to ROS by up-regulation of a transgene for an antioxidant protein, responded to cocultivation with irradiated bone marrow stromal cells. Bone marrow stromal cell line GPIa/GBL, derived from long-term bone marrow culture of a C57BL/6J-GPIa mouse, was irradiated in vitro and then cocultured with the interleukin (IL)-3-dependent hematopoietic progenitor cell line 32D cl 3, or with each of several subclonal lines expressing a transgene for human manganese superoxide dismutase (MnSOD). Cobblestone island formation, as a measure of adherence and proliferation by 32D-MnSOD clones in the presence or absence of IL-3, was increased with irradiated compared to control GPIa cells. Furthermore, using a fluorescent dye which detects ROS, hematopoietic cells cocultivated with irradiated stromal cells demonstrated higher levels of intracellular ROS than cells cocultivated and forming cobblestone islands on nonirradiated stromal cells. Since ROS are known to induce mutations in hot spots in the p53 gene, it appears worthwhile to investigate a potential mechanism for irradiated stromal cell induction of hematopoietic stem cell transformation through ROS-induced mutations. The present cell culture and molecular biology techniques provide new methods to analyze the effects of irradiated stromal cells on closely attached hematopoietic stem cells during irradiation leukemogenesis.