Early G2/M checkpoint failure as a molecular mechanism underlying etoposide-induced chromosomal aberrations
Shinichiro Nakada, Yoko Katsuki, Issei Imoto, Tetsuji Yokoyama, Masayuki Nagasawa, Johji Inazawa, Shuki Mizutani
Shinichiro Nakada, Yoko Katsuki, Issei Imoto, Tetsuji Yokoyama, Masayuki Nagasawa, Johji Inazawa, Shuki Mizutani
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Research Article Oncology

Early G2/M checkpoint failure as a molecular mechanism underlying etoposide-induced chromosomal aberrations

Abstract

Topoisomerase II (Topo II) inhibitors are cell cycle–specific DNA-damaging agents and often correlate with secondary leukemia with chromosomal translocations involving the mixed-lineage leukemia/myeloid lymphoid leukemia (MLL) gene on chromosome 11 band q23 (11q23). In spite of the clinical importance, the molecular mechanism for this chromosomal translocation has yet to be elucidated. In this study, we employed 2-color FISH and detected intracellular chromosomal translocations induced by etoposide treatment. Cells such as ataxia-telangiectasia mutated–deficient fibroblasts and U2OS cells, in which the early G2/M checkpoint after treatment with low concentrations of etoposide has been lost, executed mitosis with etoposide-induced DNA double-strand breaks, and 2-color FISH signals located on either side of the MLL gene were segregated in the postmitotic G1 phase. Long-term culture of cells that had executed mitosis under etoposide treatment showed frequent structural abnormalities of chromosome 11. These findings provide convincing evidence for Topo II inhibitor–induced 11q23 translocation. Our study also suggests an important role of the early G2/M checkpoint in preventing fixation of chromosomal abnormalities and reveals environmental and genetic risk factors for the development of chromosome 11 translocations, namely, low concentrations of Topo II inhibitors and dysfunctional early G2/M checkpoint control.

Authors

Shinichiro Nakada, Yoko Katsuki, Issei Imoto, Tetsuji Yokoyama, Masayuki Nagasawa, Johji Inazawa, Shuki Mizutani

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Figure 3

FISH analysis for chromosome 11.

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FISH analysis for chromosome 11. (A) Representative image of metaphase A...
(A) Representative image of metaphase ATM-deficient fibroblasts hybridized with the CH11C probe (green) and MLL probes (green and red overlap). Chromosomes were stained by DAPI. The arrows indicate overlapping MLL signals. (B) Representative image of metaphase ATM-deficient fibroblasts hybridized with probes for whole chromosome 11 (red) and MLL (green and red overlap). (C) Flow diagram of the cell fractionation procedure. (D) Etoposide-treated postmitotic G1 phase ATM-deficient fibroblasts with 1 (upper panel) and 3 (lower panel) centromeric 11q23 signals. (E) Proportion of metaphase and postmitotic G1 phase cells with gain (red) and loss (blue) of CH11C (left) or centromeric 11q23 (right) probe signals. n > 250 for each. *Odds ratio (OR) for etoposide treatment. **P = 0.77 and †P < 0.0001, differences in OR between postmitotic G1 and metaphase cells when cells were hybridized with CH11C and centromeric 11q23 probes, respectively (P for interaction term of cell cycle phase × treatment by a logistic regression model). (F) Micronuclei containing centromeric 11q23 signals (arrows). Cells were hybridized with centromeric 11q23 probes. Original magnification of FISH images, ×600.