REGULATED PROTEIN WHICH STIMULATES C-MYC EXPRESSION

这项建议的总体目标是通过以下方式分析机制(S) C-myc原癌基因的转录维持在 未分化的白血病细胞和终末期不可逆转的关闭 差异化。前单核细胞白血病细胞株HL-60和 单核细胞系U-937可被诱导分化 各种试剂，如二甲基亚砜，非常适合研究 这个问题。因为c-myc的存在对维护至关重要 在这些细胞中处于增殖、非终末分化状态， 阐明导致永久性减少的机制 该基因的表达在卵巢癌的发生发展中具有重要意义。 可能用于某些肿瘤治疗的生物修饰剂。 在我的博士后研究期间，我发现了一种新的核蛋白 它存在于未分化的白血病细胞中，并在体外结合 C-myc P1启动子上游1500个碱基对。这一因素在中国的约束力 Vivo到其同源序列增强嵌合c-myc的表达 CAT基因增加了约80%。重要的是，该因子的结合活性 在致力于分化的情况下消失(在细胞提取物中 用二甲基亚砜处理)，与未改变的构成相反 在此过程中，许多其他c-myc调控因子的结合活性 进程。 最近，我们从HL-60细胞中纯化了这种反式激活因子 提取并进行了一些多肽的序列分析 由酶和化学裂解产生。有了这个信息，我们现在 建议利用简并池获得全长cdna克隆 寡核苷酸探针与HL-60细胞cDNA文库杂交。 或者，双链同源序列将被用作探针 在诱导产生融合蛋白的噬菌体斑块上。有了这个克隆人，我们 将能够分析这种蛋白质的分子相互作用 与DNA结合和c-myc的反式激活有关。通过使用 Cdna探针和特异性抗体，将进行实验以 确定该因子的结合活性是否在 分化的发生是因为它的合成或调节的变化 修改。此外，其结合活性与c-myc的连接 表达和对细胞分化的研究将通过其选择性 从未分化的细胞中去除(通过转基因反义 寡核苷酸)。反之，反式激活因子将会结束- 在二甲基亚砜诱导分化的细胞中表达 确定它的存在是否会对减少碳排放产生影响 MYC转录启动和对分化的承诺。这些 研究将阐明一种新的调控机制，它调节 一种在许多肿瘤细胞的生长中起关键作用的基因。

The broad objective of this proposal is to analyze the mechanism(s) by which the transcription of the c-myc proto-oncogene is maintained in undifferentiated leukemia cells and irreversibly shut off during terminal differentiation. Both the promonomyelocytic leukemia cell line HL-60 and the monoblastic cell line U-937 can be induced to differentiate by a variety of agents, such as dimethyl sulfoxide, and are well suited to study this question. Since the presence of c-myc is critical for the maintenance of a proliferating, nonterminally differentiated state in these cells, an elucidation of mechanisms which lead to a permanent reduction in the expression of this gene has significant implications in the development of biological modifiers that might be used therapeutically for certain tumors. During my post-doctoral fellowship I discovered a novel nuclear protein that is present in undifferentiated leukemia cells and which binds in vitro 1,500 bp upstream of the c-myc P1 promoter. The binding of this factor in vivo to its cognate sequence potentiates expression from a chimeric c-myc CAT gene by approximately 80%. Importantly, binding activity of the factor disappears with commitment to differentiation (in extracts from cells treated with dimethyl sulfoxide), in contrast to the unaltered constitutive binding activity of a number of other c-myc regulatory factors during this process. Recently we have purified this transactivating factor from HL-60 cell extracts and have performed sequence analysis of a number of peptides generated by enzymatic and chemical cleavage. With this information we now propose to obtain a full-length cDNA clone using pools of degenerate oligonucleotide probes to hybridize with an HL-60 cell cDNA library. Alternatively, the double-stranded cognate sequence will be used as a probe on phage plaques induced to produce fusion proteins. With this clone we will be able to analyze molecular interactions of this protein that are associated with DNA binding and transactivation of c-myc. With the use of cDNA probes and specific antibodies, experiments will be performed to determine whether loss of the factor's binding activity during differentiation occurs because of a change in its synthesis or a modulatory modification. In addition, the linkage of its binding activity to c-myc expression and to cellular differentiation will be studied by its selective removal from undifferentiated cells (via transfected anti-sense oligonucleotides). Conversely, the transactivating factor will be over- expressed in cells induced to differentiate with dimethyl sulfoxide to determine whether its presence will have an impact on the reduction of c- myc transcriptional initiation and commitment to differentiation. These studies will elucidate a new control mechanism that regulates expression of a gene that plays a critical role in the growth of many neoplastic cells.