Early G2/M checkpoint failure as a molecular mechanism underlying etoposide-induced chromosomal aberrations
Shinichiro Nakada, … , Johji Inazawa, Shuki Mizutani
Shinichiro Nakada, … , Johji Inazawa, Shuki Mizutani
Published January 4, 2006
Citation Information: J Clin Invest. 2006;116(1):80-89. https://doi.org/10.1172/JCI25716.
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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 2

ATM-deficient fibroblasts and U2OS cells execute mitosis in the presence of low-dose etoposide.

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ATM-deficient fibroblasts and U2OS cells execute mitosis in the presence...
(A) DNA content as analyzed in ATM-deficient fibroblasts after treatment with the indicated concentrations of etoposide for 16 hours. The percentage of cells at S and G2/M phases is indicated. (B and C) Percentages of M phase cells as analyzed before and after exposure to etoposide together with colcemid for 3 hours in normal and ATM-deficient fibroblasts. (B) Representative data analyzed by flow cytometry are shown. Dots representing M phase cells are enclosed within the squares. (C) The relative increment of the percentage of M phase cells under etoposide treatment was calculated against that under mock treatment. The abscissa is logarithmic (0 is shown separately). Data in the graph represent mean ± SD of 3 separate experiments. (D) Flow cytometric analysis for the M phase fraction of ATM-deficient fibroblasts. Cells were harvested soon after shake-off and incubated for an additional 2 hours with 0 or 4 μM etoposide. (E) The MOI, calculated as described in Methods. DNA content was analyzed in the BrdU-negative fraction of normal and ATM-deficient fibroblasts after 1- or 5-hour treatment. (F and G) The relative MOI in the presence of etoposide calculated against MOI in its absence. Cells were treated with the indicated concentrations of etoposide. Data represent mean ± SD of 3 separate experiments. (F) Relative MOI of normal ATM-deficient fibroblasts and U2OS cells. The abscissa is logarithmic (0 is shown separately). (G) Relative MOI of ATM-deficient fibroblasts with (pEBSYZ5) and without (pEBS7) ATM complementation.