Mutator Phenotype in Human Cancers

描述（由申请人提供）：本提案的目的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重突变，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自己的基因组，只有一个或最多几个错误。相比之下，癌细胞表现出大量的染色体畸变，我们假设它们可能包含数百到数千个额外的DNA序列随机变化。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入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分层，提供肿瘤异质性指标，预测恶性和转移潜力以及治疗结果。我们已经建立了一个分析，可以检测一个核苷酸替代时，存在于1亿个正确的核苷酸。我们的方案使我们能够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。我们将使用我们的实验来检查不同正常人类组织、不同人类癌症和培养的人类细胞的核DNA中的核苷酸变化。我们将测量前列腺癌中线粒体DNA的变化与肿瘤分级的关系。如果突变频率的增加驱动肿瘤进展，那么确定能够抑制突变的药物从而预防或阻碍人类癌症的进展是极其重要的。

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.