MUTATIONAL LESIONS SPECIFIC FOR IONIZING RADIATION

电离辐射特有的突变损伤

基本信息

批准号：
3200791
负责人：
HOWARD L LIBER
金额：
$ 20.12万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-04-15 至 1995-03-31
项目状态：
已结题

项目摘要

During the course of experiments to define the spectrum of X-ray-induced mutations at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus in human lymphoblastoid cells, a complex mutation was identified. This mutation has a six base-pair deletion 3 base-pairs downstream of a base-pair substitution. The same mutation has appeared 3 times independently. Based on the number of mutants examined so far, it has been calculated that this particular mutation may constitute as much as 10% of all X-ray-induced events at hprt. However, this alteration has never been observed in more than 500 other mutants examined that arose either spontaneously or after induction by other agents. Consequently, this specific mutation may be a distinctive marker for radiation exposure. It is proposed to develop the technology for the rapid and facile detection of this mutation and then to validate its use in several systems, including human cells in culture, peripheral lymphocytes from cancer patients treated with radiation, and splenic lymphocytes from irradiated mice. Furthermore, the proposed work will seek to identify other X-ray specific mutations in the in vivo mouse model and in the cancer patients. Specific Aim 1 is to develop molecular techniques for the rapid detection and quantification of this X-ray-specific mutation in populations of mixed hprt- mutants of human cells. Two potential approaches will be evaluated. The first is based on a polymerase chain reaction (PCR) assay utilizing a primer which hybridizes specifically to the mutant region. The second is based on differential migration of mutant sequences in denaturing gradient gels. Aim 2 is to examine the dose-response relationship for the induction of this mutation in human lymphoblast cells treated in vitro with radiation, by use of the techniques developed in Aim 1. The third Specific Aim is to determine whether this mutation is induced in an in vivo mouse mutation assay, recently developed by Dr. Skopek. If the X-ray specific mutant is formed in the mouse hprt gene, the dose-response relationship of its induction will be examined. Also, an approach will be developed to look for other mutations specifically induced in vivo by X-rays, based on analyses of mixed mutant populations by denaturing gradient gel electrophoresis. Finally, Specific Aim 4 is to determine whether the X-ray specific mutation is induced and persists in cancer patients who have been treated with high doses of ionizing radiation. Some Hodgkin's disease patients exhibit persistently elevated mutant frequencies many months after the end of treatment, while others have normal frequencies. These two subsets, as well as normal healthy adults, will be compared. Mutant DNAs from these patients also will be screened for additional X-ray specific mutations, by use of the protocols developed in Aim 3.

在定义 X 射线诱导光谱的实验过程中次黄嘌呤鸟嘌呤磷酸核糖转移酶 (hprt) 突变在人淋巴母细胞的位点上，鉴定出一个复杂的突变。该突变有 6 个碱基对缺失 3 个碱基对下游碱基对取代。相同突变出现3次独立。根据迄今为止检查的突变体数量，据计算，这种特殊突变可能占 10% HPRT 时所有 X 射线诱发的事件。然而这种改变从未被在 500 多个其他突变体中观察到，这些突变体要么出现自发地或在其他物质诱导后。因此，这特定突变可能是辐射暴露的独特标志。它建议开发快速简便的检测技术这种突变，然后验证其在多个系统中的使用，包括培养的人类细胞、接受治疗的癌症患者的外周淋巴细胞辐射和来自受辐射小鼠的脾淋巴细胞。此外，拟议的工作将寻求识别其他 X 射线特异性突变体内小鼠模型和癌症患者。具体目标 1 是开发用于快速检测的分子技术以及混合群体中这种 X 射线特异性突变的量化 hprt-人类细胞的突变体。将评估两种潜在的方法。第一个是基于聚合酶链式反应 (PCR) 测定，利用与突变区域特异性杂交的引物。第二个是基于突变序列在变性梯度中的差异迁移凝胶。目标 2 是检查诱导的剂量-反应关系体外处理的人淋巴母细胞中的这种突变辐射，通过使用目标 1 中开发的技术。第三个具体目的是确定这种突变是否是在小鼠体内诱导的突变检测，由 Skopek 博士最近开发。如果 X 射线具体小鼠hprt基因中形成突变体，剂量反应关系将检查其感应。此外，还将制定一种方法寻找 X 射线在体内特异性诱导的其他突变，基于通过变性梯度凝胶分析混合突变体群体电泳。最后，具体目标 4 是确定 X 射线是否特定的突变在接受过癌症治疗的癌症患者中被诱导并持续存在经高剂量电离辐射处理。某些霍奇金病患者在数月后表现出持续升高的突变频率治疗结束时，其他频率正常。这两个将比较亚群以及正常健康成年人。突变DNA 还将对这些患者进行额外的 X 射线特异性筛查突变，通过使用目标 3 中开发的方案。