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. 2009 Sep;20(17):3953-64.
doi: 10.1091/mbc.e09-01-0022. Epub 2009 Jul 8.

Divergent S phase checkpoint activation arising from prereplicative complex deficiency controls cell survival

Eric Lau  1 , Gary G Chiang, Robert T Abraham, Wei Jiang
Affiliations

Divergent S phase checkpoint activation arising from prereplicative complex deficiency controls cell survival

Eric Lau et al. Mol Biol Cell. 2009 Sep.

Abstract

The DNA replication machinery plays additional roles in S phase checkpoint control, although the identities of the replication proteins involved in checkpoint activation remain elusive. Here, we report that depletion of the prereplicative complex (pre-RC) protein Cdc6 causes human nontransformed diploid cells to arrest nonlethally in G1-G1/S and S phase, whereas multiple cancer cell lines undergo G1-G1/S arrest and cell death. These divergent phenotypes are dependent on the activation, or lack thereof, of an ataxia telangiectasia and Rad3-related (ATR)-dependent S phase checkpoint that inhibits replication fork progression. Although pre-RC deficiency induces chromatin structural alterations in both nontransformed and cancer cells that normally lead to ATR checkpoint activation, the sensor mechanisms in cancer cells seem to be compromised such that higher levels of DNA replication stress/damage are required to trigger checkpoint response. Our results suggest that therapy-induced disruption of pre-RC function might exert selective cytotoxic effects on tumor cells in human patients.

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Figures

Figure 1.
Figure 1.
Depletion of Cdc6 results in nonlethal cell cycle block in nontransformed cells, but cell cycle arrest and cell death in cancer cells. (A–F) Indicated cell types were transfected with 100 nM siLuc or siCdc6 for 72 h. Transfected cells were fixed and stained with PI, followed by FACS analysis of >10,000 cells per condition. Sub-G1 populations were calculated based upon all counts per sample; G1/S/G2/M populations were calculated from non–sub-G1 counts. Below each FACS profile, siRNA-treated cells were lysed and subjected to immunoblotting analysis with anti-Cdc6 or anti-GAPDH (loading control) antibody. Note: It is not possible to strictly distinguish G1 and early S phase cells or late S phase cells and G2/M cells by FACS analysis. Thus, G1 contains G1 and G1/S cells and G2/M contains late S and G2/M cells.
Figure 2.
Figure 2.
Nonlethal cell cycle block induced by Cdc6 deficiency in RPE1 cells requires ATR. (A) Asynchronous RPE1 cells were treated with siLuc or siCdc6 for 60 h; with siLuc or siCdc6 for 48 h and then 2.5 mM caffeine (CAFF) for 12 h; or with siLuc or siCdc6 for 12 h and then with siATR for 48 h, as indicated. Treated cells were harvested and subjected to FACS. Sub-G1 populations were calculated based upon all counts per sample; G1/S/G2/M populations were calculated from non–sub-G1 counts. As mentioned in Figure 1, G1 contains G1 and G1/S cells and G2/M contains late S and G2/M cells. (B) Cells treated as described in A were subjected to immunoblotting analysis with indicated antibodies.
Figure 3.
Figure 3.
Cdc6 deficiency induces distinct G1-G1/S arrest and activation of ATR-dependent S phase checkpoint signaling required for S phase arrest in RPE1 cells. (A) G0 serum deprivation-synchronized RPE1 cells (1) were stimulated with 10% fetal bovine serum (FBS) and immediately transfected with 200 nM siLuc (2), 20 nM siCdc6 (3), 100 nM siCdc6 (4), or 200 nM siCdc6 (5). Twenty hours after transfection, cells were harvested and subjected to FACS. G0-synchronized RPE1 cells were stimulated with 10% FBS and transfected with 200 nM siLuc (6), 20 nM siCdc6 (7), 100 nM siCdc6 (8), or 200 nM siCdc6 (9). Forty hours after transfection, cells were harvested and subjected to FACS. G0-synchronized cells as in 1 or 6–9 were subjected either to additional 40 h of serum deprivation (10) or to additional 20 h of transfection + 2.5 mM caffeine (11–14) and were harvested and analyzed by FACS. Sub-G1 populations were calculated based upon all counts per sample; G1/S/G2/M populations were calculated from non–sub-G1 counts. As mentioned in Figure 1, G1 contains G1 and G1/S cells and G2/M contains late S and G2/M cells. (B) Whole-cell lysates (left) or chromatin-bound lysates (right) from the G0-synchronized RPE1 cells subjected to indicated conditions as described in A were immunoblotted with indicated antibodies. Note: Faster migrating band observed in anti-MCM2 blots comprises phosphorylated MCM2 species (Tsuji et al., 2006).
Figure 4.
Figure 4.
S phase arrest induced by Cdc6 deficiency in nontransformed cells results from ATR-dependent DNA replication inhibition. Asynchronously growing RPE1 cells were transfected with indicated siRNA for 72 h. Transfected cells were pulse labeled with 20 μM BrdU for 15 min, fixed, CSK extracted and immunostained with indicated antibodies and DAPI (DNA). Representative nuclei from each siRNA treatment condition are shown.
Figure 5.
Figure 5.
Cdc6 deficiency in nontransformed cells results in ATR-mediated checkpoint suppression of progressing forks. (A) Dual-labeled (green, CldU and red, IdU) DNA fibers were generated from asynchronously growing RPE1 cells treated with indicated siRNA(s). Representative labeled DNA fibers and percentage of total DNA fibers for new firing origins (NFO) or progressing forks (PF) for each condition are shown. We counted ≥950 labeled DNA fibers from three independent experiments for each condition. (B) Lengths of all counted replication forks were measured by ImageJ (National Institutes of Health), sorted into increasing 3-μm bins, and plotted as percentage of total fork histograms on Excel (Microsoft). (C) Histogram of increased bin resolution (0.5-μm bins) showing a more detailed replication fork length distribution of siCdc6- and siATR-cotreated RPE1 cells.
Figure 6.
Figure 6.
HeLa and HCT116 cancer cells do not completely inhibit replication fork progression in response to Cdc6 deficiency. Dual-labeled (green, CldU and red, IdU) DNA fibers were generated for asynchronously growing HeLa (A) or HCT116 (B) cells treated with indicated siRNA(s). Representative labeled DNA fibers and percentage of total labeled DNA fibers for new firing origins (NFO) or progressing forks (PF) for each condition are shown. We counted ≥950 labeled DNA fibers from three independent experiments for each condition. Lengths of all counted replication forks were measured as described in Figure 4, B and C.
Figure 7.
Figure 7.
Cdc6 deficiency alters HP1 localization and increases γ-H2AX staining in HeLa and HCT116 cancer cells in the presence of aphidicolin. Asynchronously growing RPE1, HeLa, or HCT116 cells were treated with indicated siRNA for 48 h (A) or with indicated siRNA and then an additional 4 h in the presence or absence of 1 μM APH (B). Cells were fixed and immunostained with indicated antibodies and DAPI (DNA). (C) Whole-cell lysates from cells treated as described in B were immunoblotted with indicated antibodies.
Figure 8.
Figure 8.
Schematic of cell cycle response to pre-RC depletion. Schematic of cell cycle response to pre-RC depletion is shown (for details, see text).
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