First published online April 1, 2009
doi: 10.1242/10.1242/jcs.041889
Journal of Cell Science 122, 1184-1191 (2009)
Published by The Company of Biologists 2009
Redundant and differential regulation of multiple licensing factors ensures prevention of re-replication in normal human cells
Nozomi Sugimoto1,2,
Kazumasa Yoshida1,
Yasutoshi Tatsumi1,3,
Takashi Yugawa1,
Mako Narisawa-Saito1,
Shou Waga2,
Tohru Kiyono1 and
Masatoshi Fujita1,*
1 Virology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuohku, Tokyo 104-0045, Japan
2 Faculty of Science, Japan Women's University, 2-8-1 Mejirodai, Bunkyouku, Tokyo 112-8679, Japan
3 Division of Biochemistry, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuohku, Chiba 260-8717, Japan
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Fig. 1. Differences in sensitivities to Cdt1-induced re-replication among three human cell lines. (A) HFF2/T, HEK 293T, and HeLa cells were infected with retroviruses expressing wild-type (WT), T29A or Cy+D1m T7-Cdt1 or with control retroviruses (MOI=1 for HeLa and HEK 293T, and MOI=10 for HFF2/T), and selected with hygromycin B. At 4 days after infection, cells were collected and DNA content was analyzed by flow cytometry. The means and SDs of the percentages of re-replicated cells (the DNA content higher than 4N) from two independent experiments are shown on the right. (B) At 4 days after infection, whole-cell lysates were immunoblotted with the indicated antibodies. Actin served as a loading control. The signal intensities of the Cdt1 proteins were quantified, normalized to the signals of actin, and shown with the endogenous Cdt1 in HFF2/T cells set at 1. The means and SDs from two independent experiments are given (lower panel).
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Fig. 2. Simultaneous deregulation of Cdt1+CDC6 or Cdt1+ORC1 induces re-replication, and triple overexpression synergistically induces strongest re-replication in HEK 293T cells. Cells were transfected with mixtures of the three different expression vectors (T7-Cdt1, Flag-ORC1, 2HA-CDC6) or their empty vectors, as indicated. In all experiments, a GFP expression vector was also included to monitor transfection efficiency. Forty-eight hours after transfection, cells were analyzed. (A,B) DNA content was analyzed by flow cytometry. In these diagrams, the x-axis is FL2-A (area) and represents whole propidium iodide (PI) signals and thus DNA content. The y-axis is FL2-W (width) and represents the duration of PI signals. Dots with higher FL2-W signals, which result from aggregated cells or cell debris, were excluded from measurement of re-replicated cells. The means and SDs of the percentages of re-replicated cells (the DNA content higher than 4N) from two independent experiments are shown in B. (C) Whole-cell lysates were prepared and subjected to immunoblotting with the indicated antibodies. GFP served as a control. The signal intensities of the phospho-ATM and phospho-Chk2 proteins were quantified and the means and SDs from two independent experiments are shown with the Cdt1 overexpression alone set at 100 (lower panel). (D) Whole-cell extracts or chromatin- and nuclear-matrix-bound fractions were prepared and immunoblotted with the indicated antibodies. The signal intensities of the chromatin-bound MCM3 and MCM7 proteins were quantified, normalized to the signals of nuclear lamin C, and shown with the value of control transfectant set at 100. The means and SDs from two independent experiments are given. *P<0.05.
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Fig. 3. The CDC6 AAA mutant deficient in Cdk phosphorylation induces stronger re-replication than the when coexpressed with Cdt1 in HEK 293T cells. Cells were transiently transfected with each of the wild-type (WT), AAA or EEE 2HA-CDC6 expression vectors, with or without wild-type T7-Cdt1. In all experiments, a GFP expression vector was also included to monitor transfection efficiency. Forty-eight hours after transfection, cells were analyzed as for Fig. 2. (A,B) DNA content was analyzed. The means and SDs of the percentages of re-replicated cells from two independent experiments are shown in B. (C) Whole-cell lysates were subjected to immunoblotting with the indicated antibodies. GFP served as a control. (D) Whole-cell extracts (T), Triton X-100-extractable fractions (S), and nuclear chromatin- and nuclear-matrix-bound fractions (P) were prepared from HEK 293T cells transfected with various 2HA-CDC6s and immunoblotted with anti-CDC6, anti-Lamin A/C (nuclear protein) and anti-Ras (cytoplasmic protein) antibodies. (E) Chromatin- and nuclear-matrix-bound fractions were also immunoblotted with anti-MCM3 and anti-MCM7 antibodies. The signal intensities of the chromatin-bound MCM3 and MCM7 proteins were quantified and normalized to signals for nuclear lamin C; they are shown with the value of the control transfectant set at 100. The means and SDs from three independent experiments are given. *P<0.05.
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Fig. 4. Simultaneous deregulation of Cdt1+CDC6 or Cdt1+ORC1 induces re-replication, and triple overexpression synergistically induces strongest re-replication in normal human HFF2/T fibroblasts. Cells were co-infected with three different recombinant retroviruses as indicated or with their control empty vectors (MOI=1 for each). Cells were then co-selected with hygromycin B and puromycin. At 4 days after infection, cells were collected and analyzed. (A,B) DNA content was analyzed by flow cytometry. The means and SDs of the percentage of re-replicated cells from two independent experiments are shown in B. (C) Whole-cell lysates were immunoblotted with the indicated antibodies. The membranes were also subjected to CBB staining to show equal loading.
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© The Company of Biologists Ltd 2009
