ROLE OF BONE-MARROW STROMAL CELLS IN IRRADIATION LEUKEMOGENESIS

The role of bone marrow stromal cells of the hematopoietic microenviro nment in ionizing-irradiation leukemogenesis is a focus of current inv estigation. Evidence from recent in vitro and in vivo experiments sugg ests that damage by slowly proliferating cells of the hematopoietic mi croenvironment contributes to the sustained survival of irradiation-da maged hematopoietic progenitor cells/stem cells and can contribute to the selection and proliferation of a malignant clone. The molecular me chanism of the interaction of irradiated stromal cells with attached h ematopoietic cells has been difficult to evaluate. Irradiated bone mar row stromal cell line D2XRII demonstrated altered patterns of fibronec tin splicing and increased expression of several transcriptional splic e variants of macrophage-colony-stimulating factor. Differential displ ay has revealed specific radiation-induced gene transcripts which pers ist after irradiation of stromal cells in vitro or in vivo. In recent experiments, we demonstrated that irradiation of mouse bone marrow str omal cell line D2XRII induces release of significant levels of transfo rming 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 trans gene for an antioxidant protein, responded to cocultivation with irrad iated bone marrow stromal cells. Bone marrow stromal cell line GPIa/GB L, derived from long-term bone marrow culture of a C57BL/GJ-GPIa mouse , was irradiated in vitro and then cocultured with the interleukin (IL )-3-dependent hematopoietic progenitor cell line 32D cl 3, or with eac h of several subclonal lines expressing a transgene for human manganes e superoxide dismutase (MnSOD). Cobblestone island formation, as a mea sure of adherence and proliferation by 32D-MnSOD clones in the presenc e or absence of IL3, was increased with irradiated compared to control GPIa cells. Furthermore, using a fluorescent dye which detects ROS, h ematopoietic cells cocultivated with irradiated stromal cells demonstr ated higher levels of intracellular ROS than cells cocultivated and fo rming cobblestone islands on nonirradiated stromal cells. Since ROS ar e known to induce mutations in hot spots in the p53 gene, it appears w orthwhile to investigate a potential mechanism for irradiated stromal cell induction of hematopoietic stem cell transformation through ROS-i nduced mutations. The present cell culture and molecular biology techn iques provide new methods to analyze the effects of irradiated stromal cells on closely attached hematopoietic stem cells during irradiation leukemogenesis.