Fuscoe J C, Setzer R W, Collard D D, Moore M M
Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711, USA.
Carcinogenesis. 1996 May;17(5):1013-20. doi: 10.1093/carcin/17.5.1013.
A t(14;18) chromosomal translocation is found in approximately 85% of follicular lymphomas by both cytogenetic and molecular analyses. This rearrangement deregulates expression of the bcl-2 proto-oncogene by translocation into the immuno-globulin heavy chain locus and is probably mediated by illegitimate V(D)J recombination. We have developed a quantitative nested PCR method for detecting this event in lymphocytes of healthy individuals. Genomic DNA is purified from peripheral blood lymphocytes, and 2.5 microg (representing 4 X 10(5) cells) are amplified with translocation-specific primers under conditions in which a single copy, if present, will give a detectable PCR product. Multiple replicates are analyzed for each individual, and Poisson statistics are then used to estimate the translocation mutant frequency. We have examined lymphocyte DNA from 34 healthy individuals by this assay and found the frequency of cells with t(14;18) to range from <0.8-96X10(-7). The molecular nature of the translocations has been investigated by determining the DNA sequence at the translocation junctions. In several individuals, multiple isolates of the same translocation event were recovered, indicating that the cell with the original translocation had undergone clonal expansion. In addition, multiple independent translocations were shown to occur within an individual. Since this translocation appears to be one step in the progression of a normal cell to a cancer cell, this assay may have utility as an effects biomarker for environmental carcinogen exposure.
通过细胞遗传学和分子分析发现，约85%的滤泡性淋巴瘤存在t(14;18)染色体易位。这种重排通过易位至免疫球蛋白重链基因座而使bcl-2原癌基因的表达失调，可能由异常的V(D)J重组介导。我们开发了一种定量巢式PCR方法，用于检测健康个体淋巴细胞中的这一事件。从外周血淋巴细胞中纯化基因组DNA，取2.5微克（相当于4×10⁵个细胞），用易位特异性引物在单拷贝（若存在）能产生可检测PCR产物的条件下进行扩增。对每个个体进行多次重复分析，然后用泊松统计法估算易位突变频率。我们用该检测方法检测了34名健康个体的淋巴细胞DNA，发现t(14;18)细胞的频率范围为<0.8 - 96×10⁻⁷。通过确定易位连接点处的DNA序列，对易位的分子性质进行了研究。在几个个体中，回收了同一易位事件的多个分离株，表明具有原始易位的细胞发生了克隆扩增。此外，还显示在一个个体内发生了多个独立的易位。由于这种易位似乎是正常细胞向癌细胞进展过程中的一个步骤，该检测方法可能作为环境致癌物暴露的效应生物标志物。
