MOLECULAR GENETIC AND CYTOGENETIC APPROACHES

神经纤维瘤病（NF）是一种常染色体显性遗传疾病， 2500人在美国，而其表现形式可以是高度 可变的，大多数受影响的个体将发展为多发性神经纤维瘤 （良性肿瘤产生的神经鞘）的成年，和弗兰克 恶性肿瘤的发生率增加，无论是由于恶性肿瘤， 既存丛状神经纤维瘤的变性或作为原发事件。 因此，这种情况属于家族性癌症综合征的范畴。 癌症分子基础研究的新进展 已经发生了识别，克隆，和细胞测序 致癌基因 NF可能是一种激活突变， 这种癌基因本身不足以诱导恶性肿瘤， 转化，但促进肿瘤形成的发展， 其他事件的发生，导致了这里提出的实验。 我们建议 使用DNA介导的基因转移来寻找激活的癌基因活性， NF. 已知NF DNA不会转化NIH 3 T3细胞，但 该系统仅检测已知癌基因的子集（ras癌基因的那些 类别）。 我们将用NF DNA转染原代大鼠胚胎成纤维细胞 再加上一个激活的ras基因来寻找其他类型的激活的癌基因 并将NF细胞作为DNA转染的受体 实验 为此，我们最近开发了高效的 分离人皮肤成纤维细胞的方法。 此外，我们还将 Southern杂交发现不同NF家族的癌基因重排 印迹和染色体分析，以及癌基因异常 通过成纤维细胞mRNA的北方印迹分析表达。 一大组人 癌基因探针将用于这些印迹实验。 还将研究NF的替代机制，其中NF 突变发生在通常抑制生长的基因中;只要 该基因的另一个等位基因是活性的，则不发生表型效应。 如果 另一个等位基因由于体细胞突变或染色体丢失而失活， 然而，不正常的增长将随之而来。 该模型已被证明 适用于视网膜母细胞瘤。 我们建议测试这个模型，并试图映射 通过仔细的神经纤维瘤细胞遗传学分析， NF患者的神经纤维肉瘤，以及通过插入 使用高效逆转录病毒载体诱变NF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.