Mutator Phenotype in Human Cancers

描述(申请人提供)：这项建议的目标是确定正常和恶性人类细胞中随机突变的频率和类型。我们的假设是，正常的突变率不足以解释在癌症中观察到的多种突变，并且癌细胞表现出突变表型。在每个分裂周期中，正常的人类细胞能够复制它们的基因组，只有一个错误，或者最多几个错误。相反，癌细胞表现出大量的染色体异常，我们推测它们可能在DNA序列中包含数百到数千个额外的随机变化。大量的随机突变(即，发生在肿瘤的一个或几个细胞中的突变)可能导致肿瘤的进展，并可能解释肿瘤内癌细胞的异质性、对化疗的快速耐药性以及癌细胞侵袭邻近组织和转移的能力。肿瘤中随机突变频率的定量可能有助于癌症的分层，提供肿瘤异质性的指标，并预测恶性和转移的潜力和治疗结果。我们已经建立了一种检测方法，当存在于1亿个正确的核苷酸中时，可以检测到一个核苷酸替换。我们的方案允许我们测量内含子和外显子以及分裂和非分裂细胞中核苷酸序列随机变化的频率。我们将使用我们的分析方法来检测不同正常人类组织、不同人类癌症以及培养中的人类细胞的核DNA的核苷酸变化。我们将测量前列腺癌线粒体DNA的变化与肿瘤分级的关系。如果突变频率的增加推动了肿瘤的进展，那么确定能够抑制突变从而防止或阻碍人类癌症进展的药物是极其重要的。

DESCRIPTION (provided by applicant): The objective of this proposal is to determine the frequency and types of random mutations in normal and malignant human cells. Our hypothesis is that normal mutation rates are insufficient to account for the multiple mutations observed in cancers and that cancer cells exhibit a mutator phenotype. Normal human cells are able to copy their genomes with only one, or at most a few, mistakes during each division cycle. In contrast, cancer cells exhibit large numbers of chromosomal aberrations and we postulate that they may harbor hundreds to thousands of additional, random changes in DNA sequence. Large numbers of random mutations (i.e., mutations that occur in one or only a few cells of a tumor) could contribute to tumor progression, and could account for the heterogeneity of cancer cells within a tumor, the rapid emergence of resistance to chemotherapy, and the ability of cancer cells to invade adjacent tissues and to metastasize. Quantitation of random mutation frequency in tumors may be useful in stratifying cancers, providing an index of tumor heterogeneity, and prognosticating malignant and metastatic potential and treatment outcome. We have established an assay that can detect one nucleotide substitution when present in one hundred million correct nucleotides. Our protocol allows us to measure the frequency of random changes in nucleotide sequence in introns and exons, and in dividing and non-dividing cells. We will use our assays to examine nucleotide changes in nuclear DNA from different normal human tissues, in different human cancers, and in human cells in culture. We will measure changes in mitochondrial DNA in prostatic cancers in relationship to tumor grade. If increases in mutation frequency drive tumor progression, it is extremely important to identify agents that can inhibit mutagenesis and thereby prevent or impede the progression of human cancers.