STEM CELL AGE AND X-RAY CHEMOTHERAPY LEUKEMOGENESIS

我们已经证明了平台期培养物的x射线 永生化克隆小鼠骨髓基质细胞系的研究 内皮表型(I型和IV型胶原和层粘连蛋白 阳性)诱导一种刺激恶性的体液因素 共培养的IL-3/GM-CSF依赖的造血细胞的转化 祖细胞是髓系白血病。被称为白血病的因素 基质因子(LSF)与分子克隆的区别 基因产物多粒细胞集落刺激因子(IL-3)、IL-1、GM-CSF、M-CSF和G-CSF。 LSF可诱导基质细胞系“回家”至下列骨髓窦 注射到受照射的小鼠体内并发挥作用。 其他克隆的骨髓基质细胞系不释放LSF。 辐射。多潜能造血干细胞的共培养 分泌LSF基质细胞的B6SUtA细胞系(IL-3依赖) 诱导不依赖于因子的、非自分泌的亚克隆 成年受体小鼠的供体来源肿瘤。高效液相色谱分离纯化 和LSF在无血清条件培养液中的特性 经X射线照射的D2XRII骨髓基质细胞系显示一种蛋白质n 用上述细胞因子的抗血清中和。兔抗血清， TO LSF是我们在lambda-gtll基因中鉴定LSF克隆的方法 文库从受辐射的LSF阳性细胞系中提取。LSF克隆将 转移到表达载体系统中用于生产 重组因子。我们现在建议：1)完成分子 LSF基因的克隆及序列分析；2)研究诱导基因 利用LSF在造血靶细胞中制备消减cDNA 文库使用LSF响应型亲本B6SUtA和LSF- 诱导非IL-3非自分泌恶性亚克隆 B6SUtA。我们将描述x射线诱导的机制和 通过详细描述LSF的可能环节发挥LSF的体外作用 DNA修复和X射线损伤相关基因的表达 细胞基因产品。我们将进行无线电标记研究，以 靶向LSF细胞内，研究结合半衰期和 LSF在靶细胞膜上的周转，并表征 新鲜骨髓基质细胞产生和靶向细胞的分类 被LSF绑定和更改。方法包括长期骨髓移植 培养，永久骨髓基质细胞系，分子 克隆和基因测序，蛋白质生物化学， 放射性碘化、细胞受体结合分析、细胞克隆和 移植和肿瘤生物学。这些研究应该进一步 阐明X射线诱发白血病的机制。

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.