Roelofs H, Tasseron-de Jong J G, van der Wal-Aker J, Rodenburg R J, van Houten G B, van de Putte P, Giphart-Gassler M
Department of Molecular Genetics, University of Leiden, The Netherlands.
Mutat Res. 1992 May;276(3):241-60. doi: 10.1016/0165-1110(92)90011-w.
Although gene amplification, a process that is markedly enhanced in tumor cells, has been studied in many different cell systems, there is still controversy about the mechanism(s) involved in this process. It is still unclear what happens to the DNA sequences that become amplified, whether they remain present at their original location (conservative gene amplification) or whether gene amplification necessarily results in a deletion at the original location (non-conservative gene amplification). We have studied gene amplification in a human osteosarcoma cell line, starting from a cell clone which contains only one copy of a plasmid integrate. Independent amplificants, originating from this clone and containing elevated plasmid copy numbers, were isolated and analyzed. Based on previous observations, encompassing the persistence of single-copy DNA sequences besides amplified DNA sequences clustered at a different location in the independent amplificants, we proposed an amplification pathway including a local duplication step and transposition of the duplicated DNA to other chromosomal positions. Now we have extended our study to more independent amplificants. We prove that the single-copy plasmid-containing chromosomes in the different amplificants and the single-copy plasmid-containing chromosome in the original parental cell clone are indeed identical, namely a translocation chromosome composed of at least three parts of which two originate from chromosomes 14 and 17. We show that the unit of amplification and the unit of the proposed transposition event are at least 1.5 Mb. We also demonstrate that the amplified DNA sequences, present at genomic locations other than the original single-copy DNA sequences, are preferentially associated with chromosome 16. We find that the amplified DNA sequences are often located at or near a site of chromosome translocation involving chromosome 16. In one cell clone we detect the amplified DNA sequences in most of the cells to be located within a complete chromosome 16 while in a minority of cells the amplified sequences are located at or near a breakpoint on a translocation chromosome 16. This indicates that this amplification region is highly unstable and frequently gives rise to translocation events.
尽管基因扩增这一在肿瘤细胞中显著增强的过程已在许多不同细胞系统中得到研究，但关于该过程所涉及的机制仍存在争议。目前仍不清楚发生扩增的DNA序列会怎样，它们是仍保留在其原始位置（保守性基因扩增），还是基因扩增必然导致原始位置的缺失（非保守性基因扩增）。我们从一个仅含有一份整合质粒拷贝的细胞克隆开始，对人骨肉瘤细胞系中的基因扩增进行了研究。分离并分析了源自该克隆且含有升高的质粒拷贝数的独立扩增子。基于先前的观察，即在独立扩增子中除了聚集在不同位置的扩增DNA序列外单拷贝DNA序列持续存在，我们提出了一种扩增途径，包括一个局部复制步骤以及将复制的DNA转座到其他染色体位置。现在我们已将研究扩展到更多独立扩增子。我们证明不同扩增子中含单拷贝质粒的染色体以及原始亲本细胞克隆中含单拷贝质粒的染色体确实是相同的，即一条由至少三部分组成的易位染色体，其中两部分源自14号和17号染色体。我们表明扩增单位和所提出的转座事件单位至少为1.5 Mb。我们还证明，存在于原始单拷贝DNA序列以外基因组位置的扩增DNA序列优先与16号染色体相关联。我们发现扩增的DNA序列常常位于涉及16号染色体的染色体易位位点处或其附近。在一个细胞克隆中，我们检测到大多数细胞中的扩增DNA序列位于一条完整的16号染色体内，而少数细胞中的扩增序列位于16号易位染色体的断点处或其附近。这表明该扩增区域高度不稳定，频繁引发易位事件。
