THERAPEUTIC USES FOR HUMAN GRANULOCYTE CSF

拟议的研究旨在建立一个实验基础， 人粒细胞集落刺激因子（hG-CSF）的用途 在恶性肿瘤患者的管理中，特别是 急性骨髓性白血病 这个因素是独一无二的，因为在 体外系统，它可以刺激正常人的生产， 小鼠骨髓细胞，并且还抑制生长（可能通过 诱导分化）的一些人类和小鼠急性髓系 白血病（AML）细胞。 最近克隆了hG-CSF基因， 它在体外的表达使得 重组因子，这是这些研究所需要的。 确定了三个主要的具体目标：1）一个新的G-CSF基因 将通过测序表征来自LD 1黑素瘤系的细胞， 它将在哺乳动物和细菌中表达 表达系统，以允许表征其蛋白质 产品，并与其他G-CSF进行比较。 2)重组人G- CSF将用于研究其抑制或刺激作用， 人AML细胞系（PLB-985，KG-1）和新鲜AML细胞系的生长 来自患者的原始细胞，作为异种移植物生长或扩散 在裸小鼠的腔室中。 支持性研究将定义 rhG-CSF在小鼠体内的药代动力学，检查可能的毒性 包括由于破骨细胞活化引起的骨质减少， 预测rhG-CSF对AML作用的体外筛选试验 体内细胞 这些后面的测试将包括体外培养， G-CSF，125 I标记的G-CSF与AML细胞的结合，癌基因 表达和CSF的自分泌产生。 之间的协同作用 rhG-CSF和其它分化剂（RA，1，25-OH-VD 3， 光神霉素、阿霉素）也将在体外和 vivo. G-CSF抗性AML亚系（PLB-2 R和KG-1a）将 通过逆转录病毒载体用G-CSF基因转染， 描述AML原始细胞中G-CSF耐药的机制。 3)G-CSF改变体内骨髓毒性的能力，以及 为了提高用于治疗癌症的细胞毒性药物的治疗指数， 此外，亦会研究白血病的治疗方法。 这些研究报告将 在裸鼠中使用两种人（PLB 985，KG-1）AML细胞系， 鼠肿瘤细胞系（L1210，Dunn骨肉瘤）。 一种光谱， 时相特异性和非特异性细胞毒性药物（MTX，ADR，CTX， AraC）将与rhG-CSF一起或在rhG-CSF之后施用于小鼠。

The proposed studies seek to establish an experimental basis for the use of human granulocyte colony-stimulating factor (hG-CSF) in the management of patients with malignancies, particularly acute myelogenous leukemia. This factor is unique in that with in vitro systems, it can stimulate production of normal human and murine myeloid cells, and also inhibit the growth (possibly by inducing differentiation) of some human and murine acute myeloid leukemia (AML) cells. The recent cloning of a gene for hG-CSF, and its expression in vitro has led to the availability of recombinant factor, which is required for these studies. Three major specific aims are identified: 1) A new G-CSF gene from the LD1 melanoma line will be characterized by sequencing, and it will be expressed in both mammalian and bacterial expression systems to allows characterization of its protein product, and comparison with other G-CSFs. 2) recombinant hG- CSF will be used to study its supperssive or stimulatory effects on growth of human AML cells lines (PLB-985, KG-1) and fresh AML blasts from patients, growing as xenografts or in diffusion chambers in nude mice. Supporting studies will define the pharmacokinetics of rhG-CSF in mice, examine possible toxicity including osteopenia due to oseoclast activiation, and develop in vitro screening assays to predict the effect of rhG-CSF, on AML cells in vivo. These latter tests will include in vitro culture with G-CSF, binding of 125I-labelled G-CSF to AML cells, oncogene expression, and autocrine production of CSFs. Synergy between rhG-CSF and other differentiating agents (RA, 1,25-OH-VD3, mithramycin, adriamycin) will also be sought both in vitro and in vivo. A G-CSF-resistance AML subline (PLB-2R and KG-la) will be transfected with the G-CSF gene via a retroviral vector to characterize the mechanisms of G-CSF resistance in AML blasts. 3) The ability of G-CSF to modify the in vivo myelotoxicity, and to improve the therapeutic index of cytotoxic drugs used in leukemia treatment will also be examined. These studies will be used both human (PLB985, KG-1) AML lines in nude mice, and murine tumor lines (L1210, Dunn osteosarcoma). A specrum of phase specific and non-specific cytotoxic drugs (MTX, ADR, CTX, AraC) will be administrated with, or after rhG-CSF to mice.