MOLECULAR GENETIC AND CYTOGENETIC APPROACHES

神经纤维瘤病是一种常染色体显性遗传病，影响1/4。 在美国有2500人，而它的表现可以高度 易变的，大多数受影响的人会发展成多发性神经纤维瘤 (源于神经鞘的良性肿瘤)成年后，坦率地说 恶性肿瘤的发生率增加，要么是由于恶性 先前存在的丛状神经纤维瘤的变性或作为主要事件。 因此，这种情况属于家族性癌症综合征的范畴。 近年来对癌症分子基础认识的快速进展 随着细胞的鉴定、克隆和测序而发生 致癌基因。核因子代表一种激活突变的可能性 这种癌基因本身不足以诱发恶性 转化，但在以下情况下有助于肿瘤的形成 其他事件的发生，导致了这里提出的实验。我们建议 利用DNA介导的基因转移寻找激活的癌基因活性 Nf.已经知道，核因子DNA不会转化NIH 3T3细胞，但 该系统仅检测已知癌基因的子集(ras的那些癌基因 类别)。我们将核因子DNA导入原代大鼠胚胎成纤维细胞 加上激活的ras基因，寻找其他类型的激活癌基因 还将使用核因子细胞作为DNA转染的受体 实验。为此，我们最近开发了高效的 转染人皮肤成纤维细胞的方法。另外，我们会看一看 Southern对不同神经纤维性疾病家系癌基因重排的研究 杂交和染色体分析，以及癌基因的异常 Northern印迹法检测成纤维细胞mRNA的表达。一个广泛的人类小组 癌基因探针将用于这些印迹实验。 还将研究一种用于NF的替代机制，在该机制中 突变发生在正常情况下抑制生长的基因中；只要 该基因的另一个等位基因是活跃的，不会产生表型效应。如果 另一个等位基因因体细胞突变或染色体丢失而失活， 然而，异常增长将随之而来。这一模型已经被展示给 适用于视网膜母细胞瘤。我们建议测试该模型并尝试映射 神经纤维瘤和神经纤维瘤的细胞遗传学分析 神经纤维瘤患者的神经纤维肉瘤，以及通过插入 利用高效逆转录病毒载体诱变成纤维细胞。

Neurofibromatosis (NF) is an autosomal dominant disorder affecting 1 in 2500 individuals in the U.S. While its manifestations can be highly variable, most affected individuals will develop multiple neurofibromas (benign tumors arising from nerve sheaths) by adulthood, and frank malignancy occurs at an increased rate, either as a result of malignant degeneration of preexisting plexiform neurofibromas or as a primary event. This condition thus belongs in the category of familial cancer syndromes. Recent rapid progress in the understanding of the molecular basis of cancer has occurred with the identification, cloning, and sequencing of cellular oncogenes. The possibility that NF represents an activating mutation in such an oncogene, which is in itself insufficient to induce malignant transformation but facilitates the development of tumor formation when other events occur, leads to the experiments proposed here. We propose to use DNA-mediated gene transfer to look for activated oncogene activity in NF. It is already known that NF DNA will not transform NIH 3T3 cells, but this system detects only a subset of known oncogenes (those of the ras category). We will transfect primary rat embryo fibroblasts with NF DNA plus an activated ras gene to look for activated oncogenes of other classes and will also use NF cells as the recipients in DNA transfection experiments. For this purpose, we have recently developed efficient methods of transfecting human skin fibroblasts. In addition, we will look for oncogene rearrangements in different families with NF by Southern blotting and chromosome analysis, and for abnormalities of oncogene expression by Northern blotting of fibroblast mRNA. A broad panel of human oncogene probes will be used for these blotting experiments. An alternative mechanism for NF will also be investigated in which the NF mutation occurs in a gene that normally acts to inhibit growth; so long as the other allele of this gene is active, no phenotypic effects occur. If the other allele is inactivated by somatic mutation or chromosome loss, however, abnormal growth would ensue. This model has already been shown to apply in retinoblastoma. We propose to test this model and attempt to map the NF locus by careful cytogenetic analysis of neurofibromas and neurofibrosarcomas from patients with NF, as well as by insertional mutagenesis of NF fibroblasts using a highly efficient retrovirus vector.