STEM CELL AGE AND X-RAY CHEMOTHERAPY LEUKEMOGENESIS

我们已经证明，对平台期培养物进行x射线照射 永久性克隆小鼠骨髓基质细胞系， 内皮细胞表型（I型和IV型胶原和层粘连蛋白 阳性）诱导体液因子，刺激恶性 共培养的IL-3/GM-CSF依赖的造血细胞转化 骨髓性白血病 称为白血病的因子 基质因子（LSF）可与分子克隆的 基因产物多CSF（IL-3）、IL-1、GM-CSF、M-CSF和G-CSF。 LSF可诱导的基质细胞系“回家”到骨髓窦， 注射到辐射受体小鼠中并在体内发挥作用。 其他克隆的骨髓基质细胞系在X-射线照射后不释放LSF。 辐照 多潜能造血干细胞的共培养 细胞系B6 SUtA（IL-3依赖性）和LSF释放基质细胞 诱导因子非依赖性、非自分泌亚克隆， 成年受体小鼠中的供体来源肿瘤。 通过HPLC纯化 和LSF在无血清条件培养基中的表征， X射线照射的D2 XRII骨髓基质细胞系显示， 被上述细胞因子的抗血清中和。 兔抗血清， LSF的目的是在一个cDNA中鉴定LSF克隆， LSF-阳性细胞系的文库。 LSF克隆将 转移到表达载体系统中， 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）诱导表达基因的研究 通过LSF在造血靶细胞中制备消减cDNA 库使用LSF响应，亲本系B6 SUtA，和LSF- 诱导的IL-3非依赖性、非自分泌、恶性亚克隆， B6SUtA。 我们将描述X射线诱导的机制， LSF在体外的作用，详细说明LSF的可能联系 与DNA修复和X射线损伤相关的表达 细胞基因产物 我们将进行放射性标记研究， 靶向细胞内LSF，研究结合的半衰期， 靶细胞膜上LSF的周转，并表征 新鲜骨髓基质细胞产生和靶细胞的类别 结合并被LSF改变。 方法包括长期骨髓 培养，永久性骨髓基质细胞系，分子 克隆和基因测序，蛋白质生物化学， 放射性碘化，细胞受体结合测定，细胞克隆和 移植和肿瘤生物学。 这些研究应进一步 阐明x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.