EFFECTS OF RADIATION ON MAMALIAN CELLS

这项研究计划的长期目标是获得知识 关于参与反应的机制， 哺乳动物细胞对辐射的敏感性，特别是它的 致癌和致突变作用。这种方法是一种多方面的 一个方面，正在调查的终端包括 诱变、恶性转化、细胞存活和 诱发染色体异常。该计划有一个双重的 目的：第一，更好地界定低水平辐射的风险 通过检查诸如总剂量、 剂量率、LET和细胞内剂量分布 突变和转化；第二，了解更多关于 这些效应的细胞和分子机制。这个 具体目标是：1)确定在癌症期间被激活的癌基因 辐射导致的恶变过程。转染腺病毒载体 DNA进入NIH 3T3细胞将定期进行 经10T1/2细胞培养后，从原代和 二次转染体将通过Southern blotting和 对特定的单抗的反应性。2)检查 不同类型电离辐射对细胞的直接致突变效应 照射后c-Ha-ras原癌基因的转染 基因在质粒载体中。3)审查涉及的因素 人类的形态转化与永生化 二倍体成纤维细胞。一个特别的重点将是研究一个 源自c-myc第二内含子的DNA片段 在一株部分致病菌中诱导特殊的形态变化 转化的细胞。我们建议确定这一推定 通过缺失分析和测序获得调控元件，以及 进一步研究了它在转型中的作用。4)分析 自发性和诱发性突变的分子结构 人TK6细胞系常染色体tk基因座。恰如其分 将获得探针和限制性内切酶片段或序列 所寻找的多态标记。分子生物学技术将 被雇用，以确定是否大规模 我们观察到的变化包括基因转换，有丝分裂 重组或多位点缺失事件和估计 他们的程度。5)对突变体进行细胞遗传学分析 克隆以确定大规模DNA是否具有结构 改变与可见的染色体异常有关。 结果将与剂量印迹的结果相关联。6)至 分析其引起的分子结构变化的光谱 不同类型的辐射，包括快中子、重离子 以及发射放射性核素的俄歇。7)继续研究 转化和转化的相对生物学效应 低剂量、低剂量率中子照射的诱变作用。8)至 探讨高LET剂量微分布的作用 俄歇对细胞内辐射生物效应的影响 发射电子的放射性核素。重点将放在诱变方面 在人TK6细胞中。

The long term goal of this research program is to gain knowledge concerning the mechanisms involved in the response of mammalian cells to radiation, with particular reference to its carcinogenic and mutagenic effects. The approach is a multi- faceted one, and the endpoints under investigation include mutagenesis, malignant transformation, cell survival and the induction of chromosomal abnormalities. The program has a dual purpose: first, to better define the risks of low-level radiation exposure by examining the role of factors such as total dose, dose-rate, LET and the intracellular distribution of dose on mutagenesis and transformation; and second, to learn more about the cellular and molecular mechanisms for these effects. The specific aims are: 1) To identify oncogenes activated during the process of malignant transformation by radiation. Transfection of DNA into NIH 3T3 cells will be carried out at regular intervals after irradiation of 10T1/2 cell cultures; DNA from primary and secondary transfectants will be examined by Southern blotting and reactivity to specific monoclonal antibodies. 2) To examine the direct mutagenic effects of various types of ionizing radiation on the c-Ha-ras proto-oncogene by transfection of the irradiated gene in a plasmid vector. 3) To examine factors involved in the morphological transformation and immortalization of human diploid fibroblasts. A particular focus will be on the study of a DNA fragment derived from the second intron of c-myc which induces specific morphologic alterations in a strain of partially transformed cells. We propose to identify this presumptive regulatory element by deletion analysis and sequencing, and furthur study its role in transformation. 4) To analyze the molecular structure of spontaneous and induced mutations at the autosomal tk locus in the TK6 human cell line. Appropriate probes will be obtained and restriction fragment or sequence polymorphic markers sought. Molecular biological techniques will be employed in order to determine whether the large-scale changes we have observed involve gene conversion, mitotic recombination or multi-locus deletion events and to estimate their extent. 5) To carry out cytogenetic analysis of mutant clones in order to determine whether large scale DNA structural changes are associated with visible chromosomal abnormalities. Results will be correlated with those of dosage blots. 6) To analyse the spectrum of molecular structural changes induced by different types of radiation including fast neutrons, heavy ions and Auger emitting radionuclides. 7) To continue studies of the relative biological effectiveness for transformation and mutagenesis of low dose, low dose-rate neutron exposure. 8) To investigate the role of microdistribution of dose of high LET radiation within the cell on its biologic effects by use of Auger electron emitting radionuclides. Emphasis will be on mutagenesis in human TK6 cells.