HISTONE-GFP FUSION PROTEIN ENABLES SENSITIVE ANALYSIS OF CHROMOSOME DYNAMICS IN LIVING MAMMALIAN-CELLS

Background: The amplification of oncogenes in cancer cells is often me diated by paired acentric chromatin bodies called double minute chromo somes (DMs), which can accumulate to a high copy number because of the ir autonomous replication during the DNA synthesis phase of the. cell cycle and their subsequent uneven distribution to daughter cells durin g mitosis. The mechanisms that control DM segregation have been diffic ult to investigate, however, as the direct visualization of DMs in liv ing cells has been precluded because they are far smaller than normal chromosomes. We have visualized DMs by developing a highly sensitive m ethod for observing chromosome dynamics in living cells. Results: The human histone H2B gene was fused to the gene encoding the green fluore scent protein (GFP) of Aequorea victoria and transfected into human He La cells to generate a stable line constitutively expressing HPB-GFP. The HPB-GFP fusion protein was incorporated into nucleosomes without a ffecting cell cycle progression. Using confocal microscopy, HPB-GFP al lowed high-resolution imaging of both mitotic chromosomes and interpha se chromatin, and the latter revealed various chromatin condensation s tates in live cells. Using HPB-GFP, we could directly observe DMs in l iving cancer cells; DMs often clustered during anaphase, and could for m chromosomal 'bridges' between segregating daughter chromosomes. Cyto kinesis severed DM bridges, resulting in the uneven distribution of DM s to daughter cells, Conclusions: The HPB-GFP system allows the high-r esolution imaging of chromosomes, including DMs, without compromising nuclear and chromosomal structures and has revealed the distinctive cl ustering behavior of DMs in mitotic cells which contributes to their a symmetric distribution to daughter cells.