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First published online 11 July 2006
doi: 10.1242/jcs.03031

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Deregulation of Cdt1 induces chromosomal damage without rereplication and leads to chromosomal instability

Virology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuohku, Tokyo 104-0045, Japan

View larger version (69K): [in a new window]   Fig. 1. Cdt1 is overexpressed in various cancer cell lines. To compare the expression levels of the replication-initiation proteins in 293T and several cervical cancer cell lines (HeLa, Caski, SiHa and C33A) with normal epithelial cell lines (HCK1, HDK1, HSAEC1, HBEC1), whole-cell lysates were prepared and immunoblotted with the indicated antibodies.

View larger version (52K): [in a new window]   Fig. 2. Transient overexpression of Cdt1 in 293T cells activates the ATM-Chk2 DNA damage checkpoint pathway without rereplication. (A) Immunofluorescence analysis of 293T cells transfected with T7-Cdt1. The 293T cells were transiently co-transfected with T7-tagged wild-type human Cdt1 (Cdt1 wt), the cyclin-binding-motif-mutated T7-Cdt1 (Cdt1 Cy) or empty vector (Vector) and a puromycin resistance gene. After selection with puromycin, cells were stained with anti-T7 antibody. (B) The indicated plasmids were transfected into 293T cells as above. Whole cell extracts were immunoblotted with the indicated antibodies. * indicates the position of Chk2; ** indicates the position of phosphorylated Chk2 (P-Chk2). Cell lysates were also prepared from 293T cells treated with 2.5 mM hydroxyurea (HU). (C) The signal intensities of the bands displayed in (B) were quantified, and are shown with Cdt1 wt taken as 100%. The mean and standard deviation of two independent experiments is shown. (D) Cell-cycle analysis of 293T cells transfected with the indicated plasmids. (E) Whole-cell extracts, Triton X-100-extractable fractions and nuclear chromatin/matrix fractions were prepared from 293T cells transfected with T7-Cdt1, and immunoblotted with anti-T7, anti-Ras (cytoplasmic protein), and anti-lamin A/C (nuclear protein) antibodies.

View larger version (45K): [in a new window]   Fig. 3. Stable overexpression of human Cdt1 in Rat-1 cells activates the ATM-Chk2 DNA damage checkpoint pathway without detectable rereplication. (A) Whole-cell lysates prepared from Rat-1 clones overexpressing wild-type T7-Cdt1 (WA4, WB1, WB3 and WB4) or T7-Cdt1 Cy (CA2, CA3, CB3 and CB4) and from parental Rat-1 cells were immunoblotted. Hydroxyurea (HU)-treated Rat-1 cells were analyzed similarly. (B) Cell-cycle analysis of the Rat-1 clones. Exponentially growing clones were analyzed at 2 months post-infection. (C) Growth rates of the Rat-1 clones. (D) Whole cell extracts, Triton X-100-extractable fractions, and nuclear chromatin/matrix fractions were prepared from WB4 cells and immunoblotted as described in Fig. 2E.

View larger version (38K): [in a new window]   Fig. 4. The ATM-Chk2 DNA damage checkpoint pathway is activated by Cdt1 overexpression in G0-, S- and G2/M-phase in Rat-1 WB4 and CB4 cells. Rat-1 WB4 cells overexpressing wild-type T7-Cdt1 (A) and CB4 cells overexpressing T7-Cdt1 Cy (B) were arrested in quiescence, released, harvested at the indicated time points, and analyzed by flow cytometry and immunoblotting. (C) Asynchronous (AS) or quiescent Rat-1 or CB4 cells were treated with BrdU for 6 hours and then double-immunostained with anti-BrdU and anti-phosphorylated ATM antibodies.

View larger version (40K): [in a new window]   Fig. 5. Introduction of Cdt1 into quiescent Rat-1 cells induces chromosomal damage and activation of ATM-Chk2 without replication. Rat-1 cells were arrested in quiescence, then infected with lentiviruses carrying either wild-type or Cy mutant T7-Cdt1 or backbone vector, and subjected to analyses after 144 hours. (A) Whole-cell lysates were immunoblotted. Asynchronous (AS) CB4 cells serve as a positive control for T7-Cdt1 expression and ATM-Chk2 activation. (B) After further treatment with BrdU for 6 hours, cells were immunostained with anti-BrdU. Asynchronous (AS) Rat-1 cells serve as a positive control for BrdU uptake. (C) Cells were double-immunostained with anti-T7 and anti-phospho-ATM antibodies.

View larger version (33K): [in a new window]   Fig. 6. Stable overexpression of human Cdt1 in normal human fibroblasts activates the ATM-Chk2 DNA damage checkpoint pathway without rereplication. HFF2 cells were infected with the high-titer wild-type T7-Cdt1, the Cy mutant T7-Cdt1 or control retroviruses and selected. At 2 weeks after infection, cells were subjected to analyses. (A) Whole cell lysates were immunoblotted. (B) The signal intensities of the bands displayed in A were quantified, and shown with Cdt1wt set at 100. (C and D) Asynchronous (AS) cells or cells arrested in quiescence were treated with BrdU for 8 hours and then double-immunostained with anti-BrdU and anti-phosphorylated ATM antibodies.

View larger version (45K): [in a new window]   Fig. 7. Deregulated Cdt1 leads to chromosomal instability in normal human fibroblasts. (A) HFF2 cells were infected with the T7-Cdt1 retroviruses as in Fig. 6, and subjected to karyotypic analysis at 2 months post-infection. We performed four independent infection experiments, and chromosome distributions included the analysis of approximately 50 metaphase spreads for each experiment. The sum of results from the four experiments is shown. For parental HFF2 cells, the analysis was performed on 100 metaphase spreads. (B) Representative results of more detailed karyotypic analysis. (Top panel) Shown are normal diploid with 46 chromosomes observed in control vector-infected cells. (Middle panels) Shown are 48 chromosomes with trisomy 5 and trisomy 10 (left) and 47 chromosomes with trisomy 10 (right) observed with wt Cdt1. (Bottom panels) Shown are 45 chromosomes with Y deficiency (left) and 48 chromosomes with trisomy 7 and trisomy 10 (right) observed with Cdt1 Cy.

View larger version (52K): [in a new window]   Fig. 8. Rereplication with reduced Cdk1 activity is induced when 293T cells that overexpress Cdt1 are arrested in M phase by nocodazole. (A) 293T cells were transfected with the indicated plasmids, selected with puromycin, and further treated with nocodazole. Whole cell lysates were then immunoblotted. (B) The DNA content of cells as in A was analyzed by flow cytometry and the percent of cells with rereplicated (>4N) DNA was calculated. The mean and standard deviation of two independent experiments is shown with representative raw data. (C,D) Suppression of ATM by shRNA reduces Chk2 phosphorylation and rereplication in Cdt1-overexpressing and nocodazole-treated 293T cells. The 293T cells were infected with retroviruses expressing ATM shRNA or control retroviruses and selected. Cells were then transfected with the indicated plasmids and treated with nocodazole. Whole cell lysates were then subjected to immunoblotting. The DNA contents of the cells were analyzed by flow cytometry and the percentage of cells with rereplicated (>4N) DNA was calculated. The mean and standard deviation of two independent experiments is shown for each experiment.