MUTAGENESIS BY THE ANTINEOPLASTIC MUSTARDS

抗肿瘤芥子的诱变作用

• 批准号: 3199707
• 负责人: WILLIAM D HENNER
• 金额: $ 15.02万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-15 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3199707
• 关键词: antineoplastics; chemical carcinogen; chlorambucil; cyclophosphamide; drug adverse effect; gene deletion mutation; gene rearrangement; genetic mapping; human subject; hypoxanthine phosphoribosyltransferase; lymphoblast; mechlorethamine; melphalan; molecular cloning; molecular oncology; mutagen testing; mutagens; neoplasm /cancer genetics; nitrogen mustard; polymerase chain reaction; tissue /cell culture

This project will determine the frequency and molecular structure of the mutations induced in human lymphocytes by the nitrogen mustard group of antineoplastic alkylating agents which are well-established as carcinogens in humans. Patients treated with these drugs have an increased risk of second malignancies particularly acute leukemia. Many of the induced leukemias are associated with characteristic chromosomal deletions and rearrangements. In spite of their wide use and established carcinogenicity, relatively less is known about the mechanism of mutagenicity for the nitrogen mustards than for many environmental carcinogens. Two complementary systems will be used to study mutations induced by these DNA-DNA cross-linking agents. In both systems the genetic locus to be studied is the human hypoxanthine-guanine- phosphoribosyltranferase (HPRT) locus. In the first system, the human lymphoblastoid cell line WIL-2 NS will be exposed in vitro to some of the clinically useful alkylating agents, primarily the nitrogen mustards, and the frequency of mutations induced at various levels of cytoxicity will be determined. The polymerase chain reaction, molecular cloning and DNA sequencing will be used to identify hprt mutations as base changes, deletions or rearrangements. Preliminary studies indicate that nitrogen mustard (mechlorethamine) induces a high frequency of large gene deletions at the HPRT locus. Moreover, within the HPRT gene, a "hotspot" for mechlorethamine-induced deletions has been identified in the region between exon4 and exon9, with 16/17 of such deletions comprising the 3' end of the gene. The nature of the sequence(s) that determines the increased frequency and unidirectionality for deletions in this region will be determined by cloning and sequencing of the mutant HPRT fragments containing the breakpoint. This sequence information will allow us to test various hypotheses as to the mechanism by which such deletions occur. In the second system, lymphocytes will be obtained from patients receiving therapy with cyclophosphamide for multiple sclerosis or chlorambucil for non-Hodgkin's lymphoma. In preliminary studies, we have identified an increased mutation frequency at the HPRT locus in lymphocytes of cyclophosphamide-treated patients. The molecular structure (base substitutions, deletion endpoints, etc.) of the mutations induced in vivo by cyclophosphamide, chlorambucil and melphalan will be analyzed. These complementary systems allow study of the frequency and structure of HPRT mutations under conditions of either in vitro or in vivo exposure. Patients, nurses, pharmacists and manufacturing workers are frequently exposed to the nitrogen mustards and to related compounds. This project should allow better characterization of the exact nature of the mutagenic events that occur in human cells exposed to the nitrogen mustards (or other environmental exposures) which may later lead to second malignancies, germ line mutations and birth defects in exposed individuals.

该项目将确定频率和分子结构 氮芥类物质在人类淋巴细胞中诱导突变 抗肿瘤烷化剂已被确认为致癌物质 在人类中。 使用这些药物治疗的患者的风险增加 第二种恶性肿瘤特别是急性白血病。 许多诱发 白血病与特征性染色体缺失有关 重新安排。 尽管它们被广泛使用并已建立 致癌性，但对其机制了解相对较少 氮芥的致突变性高于许多环境 致癌物质。 两个互补系统将用于研究突变 由这些 DNA-DNA 交联剂诱导。 在这两个系统中，遗传 要研究的位点是人次黄嘌呤-鸟嘌呤- 磷酸核糖基转移酶 (HPRT) 位点。 在第一个系统中，人类 类淋巴母细胞系 WIL-2 NS 将在体外暴露于一些 临床上有用的烷化剂，主要是氮芥，和 在不同细胞毒性水平下诱导的突变频率将是 决定。 聚合酶链式反应、分子克隆和DNA 测序将用于识别 hprt 突变作为碱基变化， 删除或重新排列。 初步研究表明氮 芥末（氮芥）会导致高频率的大基因缺失 在HPRT位点。 此外，在 HPRT 基因内，有一个“热点” 已在以下区域发现了氮芥诱导的缺失： 外显子 4 和外显子 9，其中 16/17 的此类缺失包含 3' 端 基因。 决定增加的序列的性质 该区域删除的频率和单向性将是 通过突变 HPRT 片段的克隆和测序确定 包含断点。 该序列信息将允许我们测试 关于这种缺失发生的机制有各种假设。 在 第二个系统将从接受治疗的患者身上获得淋巴细胞 使用环磷酰胺治疗多发性硬化症或使用苯丁酸氮芥治疗 非霍奇金淋巴瘤。 在初步研究中，我们已经确定了 淋巴细胞 HPRT 位点突变频率增加 接受环磷酰胺治疗的患者。 分子结构（碱基 体内诱导的突变的替换、删除终点等） 将分析环磷酰胺、苯丁酸氮芥和美法仑。 这些 互补系统允许研究 HPRT 的频率和结构 体外或体内暴露条件下的突变。 患者、护士、药剂师和制造工人经常 暴露于氮芥和相关化合物。 这个项目 应该可以更好地表征诱变的确切性质 暴露于氮芥（或其他物质）的人体细胞中发生的事件 环境暴露），随后可能导致第二种恶性肿瘤、细菌 暴露个体的细胞系突变和出生缺陷。