EGF RESPONSIVE GENES WHICH REGULATE TUMOR CELL GROWTH

表皮生长因子(EGF)和其他 多肽激素对细胞生长控制的影响尚不清楚。 大多数分离和鉴定EGF反应基因的努力已经 重点放在EGF反应的早期阶段。然而， 最早的反应不足以引起增长反应 由于EGF必须持续存在较长时间 (超过6-12小时)在承诺发生之前。在这段时间里 这些细胞经历了大量的代谢变化。它有 事实证明，很难区分哪些事件涉及 从承诺所需的细胞激活，或 刺激细胞分裂。为了避免这个问题，从基因上讲 生长反应不同的相关克隆细胞系 EGF(刺激、无反应或抑制)将被用来分离 EGF反应基因的cDNA。尽管生物反应 对EGF在每种细胞类型中的早期反应是不同的 EGF诱导的克隆在差异反应中相似 细胞系；例如表皮生长因子受体在结合方面是相似的。 与EGF的亲和力，引发酪氨酸磷酸化的程度 由激素和受体的速度和程度决定的 内部化。具有代表性的大分子lambda gt10基因 用每个细胞克隆合成的cdna构建文库 在定义的时间间隔内使用EGF处理的类型。图书馆将是 消减杂交制备的cDNAs探针筛选 以丰富每个细胞的EGF反应的特异性序列 克隆类型。将使用消减杂交cdna探针 以丰富可归类的EGF响应序列 如下：1)在每种克隆细胞类型中唯一表达的那些， 2)丰富度不同的，以及3)不同的 以类似的方式监管，以回应EGF。一种比较 在EGF反应基因的类别中提供了一种开始 对监管中的重要内容进行系统分析 对细胞增殖的影响。对基因克隆的仔细分析 将被用来区分EGF调节的基因 被刺激的、被抑制的或无反应的细胞所特有的 EGF。当所需类型的候选cdna克隆 获取后，它们将用于检查正常和其他 转化的细胞对EGF有反应。有了这样的探测器， 将启动研究以识别编码基因并 考察表皮生长因子调节其功能的机制(S) 在正常细胞和转化细胞中均有表达。

The mechanism by which epidermal growth factor (EGF) and other peptide hormones influence cellular growth control are unknown. Most efforts to isolate and identify EGF responsive genes have focused on the early phases of the EGF response. However, the earliest responses are not sufficient to elicit a growth response since EGF must be present continuously for a prolonged period (over 6-12 hours) before commitment occurs. During this time the cells undergo a large number of metabolic changes. It has proven difficult to distinguish those events which are involved in cellular activation from those required for commitment to, or stimulation of cell division. To avoid this problem, genetically related cloned cell lines which differ in their growth response to EGF (stimulated, unresponsive or inhibited) will be used to isolate cDNAs to EGF responsive genes. Although the biological response to EGF is different in each cell type, many of the early responses elicited by EGF are similar in the differentially responsive cloned cell lines; eg. the EGF-receptors are similar in their binding affinity for EGF, the extent of tyrosine phosphorylation elicited by the hormone and the rate and extent of receptor internalization. Large and representative lambda gt10 cDNA libraries were made with cDNA synthesized from each cell clone type treated with EGF for defined intervals. The libraries will be screened with cDNA probes prepared by subtractive hybridization to enrich for sequences specific to the EGF response of each cell clone type. Subtractive hybridization cDNA probes will be used to enrich for EGF responsive sequences which can be categorized as follows: 1) those uniquely expressed in each cloned cell type, 2) those which differ in abundance, and 3) those which are regulated in a similar manner in response to EGF. A comparison of the classes of EGF responsive genes provide a means to begin a systematic analysis of those which are important in the regulation of cellular proliferation. A careful analysis of the cDNA clones will be used to distinguish the EGF regulated genes which are specific to cells stimulated by, inhibited by or non-responsive to EGF. When candidate cDNA clones of the desired types are obtained, they will be used to examine normal and other transformed cells responding to EGF. With such probes in hand, studies will be initiated to identify the encoded genes and to examine the mechanism(s) by which EGF regulates their expression in both normal and transformed cells.