REGULATION OF LUNG CANCER GROWTH

细胞周期进程的调节在肿瘤中被破坏， 转化的细胞，通常是因为控制细胞周期的蛋白质， 不正常地表达或者激活。这项建议的目的是 鉴定控制小细胞中细胞周期进程蛋白质 肺癌（SCLC）是一种高度转移性肿瘤， 美国每年有25,000人死亡。 一个独特的方法将是 用来识别这些蛋白质，基于我的发现， M3毒蕈碱型乙酰胆碱受体（mAChR）抑制细胞周期 在SCLC细胞中的进展。 细胞周期调节蛋白将是 有两个标准区分。 首先，蛋白质必须被修饰（通过 转录、翻译或翻译后加工的变化） 当SCLC细胞在细胞周期中进展时。 二是这些 当细胞周期进程受到以下因素的抑制时， mAChR激活。 这种新的方法也可以识别致癌的 SCLC细胞中的蛋白质。mAChR刺激可以独特地抑制SCLC 增殖，因为它使异常表达正常化， 激活有助于SCLC转化的蛋白质。 为 例如，mAChR刺激可以诱导以下的表达或激活： 在SCLC中低表达或失活的肿瘤抑制蛋白 细胞 这些受影响的蛋白质的表征可能有助于阐明 有助于SCLC转化的过程。 经历细胞周期依赖性转录后的蛋白质类 被mAChR激活抑制的修饰将被检测到， 它们在循环SCLC细胞中独特的35 S或32 P放射性标记， 到静止或用卡巴胆碱（mAChR）处理的细胞， 激动剂）。 这些蛋白质将使用针对 针对已知的调节蛋白和通过蛋白质微序列 分析. 北方印迹和核溢流转录测定将 鉴定其细胞周期依赖性积累 由mAChR激活而改变。 为了加快寻找监管机构 蛋白质，Southern印迹分析将用于鉴定蛋白质 不能参与mAChR介导的生长抑制，因为 基因缺失或重排。 曾经潜在的调节蛋白 抗体和寡核苷酸探针将在 为了比较所鉴定的蛋白质的表达和活性， 在小细胞肺癌和其他组织中。 独特的表达或激活这些 与其他细胞类型相比，SCLC细胞中的蛋白质将提供 令人信服的证据表明，已鉴定的蛋白质有助于 小细胞肺癌的肿瘤转化。

The regulation of cell cycle progression is disrupted in neoplastically transformed cells, often because proteins controlling the cell cycle are abnormally expressed or activated. The objective of this proposal is to identify proteins which control cell cycle progression in small cell lung carcinoma (SCLC), a highly metastatic tumor which causes over 25,000 deaths per year in the United States. A unique approach will be used to identify these proteins, based on my finding that activation of M3 muscarinic acetylcholine receptors (mAChR) inhibits cell cycle progression in SCLC cells. Cell cycle regulatory proteins will be distinguished by two criteria. First, the proteins must be modified (by changes in transcription, translation, or posttranslational processing) when SCLC cells progress through the cell cycle. Second, these modifications must not occur when cell cycle progression is inhibited by mAChR activation. This novel approach may also identify oncogenic proteins in SCLC cells. mAChR stimulation may uniquely inhibit SCLC proliferation because it normalizes the aberrant expression or activation of proteins contributing to SCLC transformation. For example, mAChR stimulation may induce the expression or activation of tumor suppressor proteins that are underexpressed or inactivated in SCLC cells. Characterization of these affected proteins may shed light on processes contributing to SCLC transformation. Proteins undergoing cell cycle-dependent, post-transcriptional modifications that are inhibited by mAChR activation will be detected by their unique 35S- or 32P-radiolabelling in cycling SCLC cells, compared to cells that are quiescent or treated with carbachol (an mAChR agonist). These proteins will be identified using antibodies directed against known regulatory proteins and by protein micro-sequence analysis. Northern blot and nuclear run-off transcription assays will identify mRNA transcripts whose cell cycle-dependent accumulation is altered by mAChR activation. To expedite the search for regulatory proteins, southern blot analysis will be used to identify proteins unable to participate in mAChR-mediated growth inhibition because of genetic deletion or rearrangement. Once potential regulatory proteins are identified, antibody and oligonucleotide probes will be made in order to compare the expression and activity of the identified proteins in SCLC and other tissues. Unique expression or activation of these proteins in SCLC cells, compared to other cell types, will provide compelling evidence that the identified proteins contribute to neoplastic transformation in SCLC.