STEM CELL AGE AND X-RAY CHEMOTHERAPY LEUKEMOGENESIS

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We have demonstrated that x-irradiation of plateau-phase cultures of permanent cloned mouse bone marrow stromal cell lines of fibro- endothelial phenotype (collagen types I and IV and laminin positive) induces a humoral factor that stimulates malignant transformation of cocultured IL-3/GM-CSF dependent hematopoietic progenitor cells myeloid leukemia. The factor termed Leukemogenic Stromal Factor (LSF) is distinguishable from molecularly cloned gene products multi-CSF (IL-3), IL-1, GM-CSF, M-CSF, and G-CSF. LSF inducable stromal cell lines "home" to marrow sinuses following injection into irradiated recipient mice and function in vivo. Other cloned marrow stromal cell lines do not release LSF after x- irradiation. Co-cultivation of multipotential hematopoietic stem cell line B6SUtA (IL-3 dependent) with LSF-releasing stromal cells induces factor-independent, non-autocrine subclones that produce donor-origin tumors in adult recipient mice. Purification by HPLC and characterization of LSF in serum-free conditioned medium from x-irradiated D2XRII marrow stromal cell line revealed a protein n neutralized by antisera to the above cytokines. Rabbit antisera, to LSF is being us to identify LSF clones in a lambda-gtll cDNA library from an irradiated LSF-positive cell line. LSF clones will be transferred to an expression vector system for production of recombinant factor. We now proposed to: 1) complete the molecular cloning of LSF and sequence the peptide; 2) study the genes induced by LSF in hemopoietic target cells preparing a subtraction cDNA library using the LSF-responsive, parent line B6SUtA, and an LSF- induced IL-3-independent, nonautocrine, malignant subclone of B6SUtA. We will characterize the mechanism of x-ray induction and action of LSF in vitro by detailing the probable link of LSF expression with that of DNA repair and x-ray damage associated cellular gene products. We will perform radiolabelling studies to target LSF intracellularly, study the half-life of binding and turnover of LSF on the target cell membrane, and characterize the classes of fresh marrow stromal cells producing and target cells binding and altered by LSF. Methods include long-term bone marrow cultures, permanent bone marrow stromal cell lines, molecular cloning and gene sequencing, protein biochemistry, radioiodinization, cell receptor binding assays, cells cloning and transplantation and tumor biology. These studies should further elucidate the mechanism of x-ray induced leukemia.