MUTAGENESIS BY THE ANTINEOPLASTIC MUSTARDS

该项目将确定的频率和分子结构的 在人淋巴细胞中由氮芥组诱导的突变 已被公认为致癌物的烷化剂 在人类身上。 接受这些药物治疗的患者， 第二种恶性肿瘤，特别是急性白血病。 许多诱导 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， 致癌性，相对较少了解的机制， 氮诱变性比许多环境 致癌物质。 两个互补的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， 待研究的位点是人次黄嘌呤-鸟嘌呤- 磷酸核糖基转移酶（HPRT）基因座。 在第一个系统中，人类 淋巴母细胞样细胞系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 在不同水平的细胞毒性下诱导的突变频率将是 测定 聚合酶链反应、分子克隆和DNA 测序将用于鉴定作为碱基变化的HPRT突变， 删除或重排。 初步研究表明， 芥子气（氮芥）诱导高频率的大基因缺失 在HPRT位点。 此外，在HPRT基因内， 已经在以下区域中鉴定了氮芥诱导的缺失： 外显子4和外显子9的缺失，其中16/17的这种缺失包含外显子3'末端， 基因 确定增加的序列的性质 频率和单向性删除在这个区域将是 通过突变HPRT片段的克隆和测序确定 包含断点。 这个序列信息将使我们能够测试 关于这种缺失发生的机制的各种假设。 在 第二个系统，淋巴细胞将从接受 环磷酰胺治疗多发性硬化症或苯丁酸氮芥治疗多发性硬化症 非霍奇金淋巴瘤 在初步研究中，我们发现了一种 淋巴细胞中HPRT基因座突变频率增加， 环磷酰胺治疗的患者。 分子结构（基础） 取代、缺失端点等）体内诱导的突变 通过环磷酰胺，苯丁酸氮芥和美法仑将进行分析。 这些 互补系统允许研究HPRT的频率和结构 在体外或体内暴露条件下的突变。 病人、护士、药剂师和制造业工人经常 暴露在氮芥和相关化合物中。 这个项目 应允许更好地表征诱变剂的确切性质 在暴露于氮霉素（或其它氮源）的人类细胞中发生的事件 环境暴露），随后可能导致第二种恶性肿瘤， 线突变和出生缺陷。

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.