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 6

Chromosomal aberrations in a mixture of stable clones of ATM-deficient fibroblasts, which had executed mitosis under etoposide treatment.

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Chromosomal aberrations in a mixture of stable clones of ATM-deficient f...
(A) Flow diagram for long-term culture procedure. (B, C, and E) Metaphase spreads hybridized with the CH11C probe (green) and MLL probes (green and red overlap). (B) One GM05849C-MD cell showing 4 CH11C signals and 2 pairs of MLL signals. (C) One GM05849C-ME1 cell showing 4 CH11C signals with only 1 of them bearing MLL signals. (D) Proportion of cells with gain and loss of CH11C or MLL signals. n = 300 for each. *OR for etoposide treatment. **P = 0.0002, difference in OR for interaction term of probe × treatment by a logistic regression model. (E) One GM05849C-ME1 cell with the MLL gene translocated to another chromosome. The inset is a magnified image of the enclosed area. (F) Percent of chromosomes negative for CH11C signal among those positive for MLL signals. Data were analyzed by multiple logistic regression. n = 400 for each. †P = 0.028 for interaction term of etoposide treatment × mitotic progression. Neither of the pairwise comparisons for etoposide treatment in asynchronous cells nor for mitotic progression in etoposide-untreated cells was statistically significant (P = 1.0). (G) Representative image of chromosome 11 translocation (arrow) in GM05849C-ME1 cells hybridized with chromosome 11 painting (red) and MLL probes (green and red overlap). (H and I) Abnormal MLL gene configuration. GM05849C-ME1 cells were hybridized with MLL probes. Chromosomal translocation of MLL BCR (H) and tandem duplication of the MLL gene (I). Original magnification for FISH images, ×600.