Induction of diplochromosomes in mammalian cells by inhibitors of topoisomerase II |
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Authors: | Adrian T Sumner |
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Institution: | (1) MRC Human Genetics Unit, Edinburgh, UK, GB |
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Abstract: | Diplochromosomes, consisting of four chromatids lying side-by-side, instead of the normal two, are produced when cells go
through two rounds of DNA replication without separation of chromatids. They are thus an indication of the failure of the
normal chromosome separation mechanism. In the present experiments, induction of diplochromosomes by inhibitors of topoisomerase
II (Topo II) was used to provide further evidence that Topo II is required for separation of daughter chromosomes. Actively
growing cultures of CHO cells were treated with Colcemid, and separated into metaphase and interphase fractions, each of which
was treated for 2 h with the Topo II inhibitor being tested. The cells were then cultivated in fresh medium without inhibitor
for periods of between 18 and 44 h, and metaphase cells once again accumulated by treatment with Colcemid. Chromosome preparations
were made in the standard way and stained with Giemsa. Up to 2,000 metaphases were counted from each culture, and the proportion
with diplochromosomes calculated. At appropriate concentrations, the Topo II inhibitors etoposide and mitoxantrone induced
substantial levels of metaphases with diplochromosomes in cultures that had been treated when the cells were in interphase
(up to 30% and 11%, respectively). Amsacrine, however, only produced a smaller proportion (4.7%) of metaphases with diplochromosomes
after a much longer culture period following treatment. All the inhibitors caused severe chromosome damage. When used to treat
metaphase cells, mitoxantrone and amsacrine only induced diplochromosomes after prolonged culture, although a small number
of diplochromosomes were seen after etoposide treatment and a shorter period of culture. Results with cells treated in metaphase
might indicate that Topo II is, in fact, not required for anaphase chromosome separation, although it is clearly important
for segregation of newly replicated DNA.
Received: 8 August 1998 / Accepted: 13 September 1998 |
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