Binomial Mitotic Segregation of <em>MYCN</em>-Carrying Double Minutes in Neuroblastoma Illustrates the Role of Randomness in Oncogene Amplification

BackgroundAmplification of the oncogene MYCN in double minutes (DMs) is a common finding in neuroblastoma (NB). Because DMs lack centromeric sequences it has been unclear how NB cells retain and amplify extrachromosomal MYCN copies during tumour development. Principal FindingsWe show that MYCN-carrying DMs in NB cells translocate from the nuclear interior to the periphery of the condensing chromatin at transition from interphase to prophase and are preferentially located adjacent to the telomere repeat sequences of the chromosomes throughout cell division. However, DM segregation was not affected by disruption of the telosome nucleoprotein complex and DMs readily migrated from human to murine chromatin in human/mouse cell hybrids, indicating that they do not bind to specific positional elements in human chromosomes. Scoring DM copy-numbers in ana/telophase cells revealed that DM segregation could be closely approximated by a binomial random distribution. Colony-forming assay demonstrated a strong growth-advantage for NB cells with high DM (MYCN) copy-numbers, compared to NB cells with lower copy-numbers. In fact, the overall distribution of DMs in growing NB cell populations could be readily reproduced by a mathematical model assuming binomial segregation at cell division combined with a proliferative advantage for cells with high DM copy-numbers. ConclusionBinomial segregation at cell division explains the high degree of MYCN copy-number variability in NB. Our findings also provide a proof-of-principle for oncogene amplification through creation of genetic diversity by random events followed by Darwinian selection.背景：神经母细胞瘤（neuroblastoma, NB）中，癌基因MYCN（oncogene MYCN）在双微体（double minutes, DMs）上的扩增是常见现象。由于双微体缺乏着丝粒序列，学界始终未能明确肿瘤发生过程中，NB细胞如何保留并扩增染色体外的MYCN拷贝。 主要研究结果：本研究发现，NB细胞中携带MYCN的双微体在间期向前期转变时，会从细胞核内部迁移至浓缩染色质的外周区域，并在整个细胞分裂过程中优先定位于染色体端粒重复序列附近。然而，双微体的染色体分离并不会因端粒核蛋白复合物（telosome nucleoprotein complex）的破坏而受影响，且在人-小鼠细胞杂交株中，双微体可顺利从人类染色质迁移至小鼠染色质，这表明双微体并不会结合人类染色体上的特定位置元件。通过对有丝分裂后期/末期细胞的双微体拷贝数进行统计，本研究发现双微体的染色体分离过程可通过二项式随机分布进行精准拟合。集落形成实验显示，相较于携带较低双微体拷贝数的NB细胞，高拷贝数（MYCN）双微体的NB细胞具有显著的生长优势。事实上，针对增殖中NB细胞群体内双微体的整体分布，我们可以通过一个数学模型轻松复现：该模型假设细胞分裂时双微体以二项式方式分离，且高双微体拷贝数的细胞具有增殖优势。 结论：细胞分裂过程中的二项式分离机制，可解释NB中MYCN拷贝数的高度异质性。本研究结果同时为“通过随机事件产生遗传多样性，再经达尔文选择实现癌基因扩增”这一过程提供了原理验证。