The impact of S- and G2-checkpoint response on the fidelity of G1-arrest by cisplatin and its comparison to a non-cross-resistant platinum(IV) analog

Abstract

Objective: Cisplatin is a DNA-damaging antitumor agent that is highly effective in treating ovarian cancer. It activates the p53/p21 pathway for its cytotoxic mode of action, but it does not induce p21-dependent cell cycle arrest in G1. Therefore, we investigated this paradox, and used the model analog DAP as a positive control for p21-dependent G1-arrest.

Methods: Studies were conducted in p53-proficient ovarian A2780 tumor cells to examine Cdk activity, cell cycle distribution and DNA damage signaling after cisplatin or DAP in combination with the mitotic inhibitor nocodazole.

Results: Cisplatin consistently induced transient S-phase arrest by inhibiting Cdk2/cyclin A complex in S-phase at 12 h and then a durable G2/M-arrest by inhibiting Cdc2/cyclin B complex at 12-18 h. These inhibitions were associated with Chk1 and Chk2 activation and resultant increase in inhibitory tyrosine phosphorylation of Cdk2 and Cdc2. Cisplatin also potently inhibited G1-phase Cdk4/cyclin D1 and Cdk2/cyclin E activities at ~18 h. In agreement, exposure of cisplatin-treated A2780, HCT-116(p53-/-) and HCT-116(p21-/-) tumor cells to nocodazole revealed limited G1-arrest that was dependent on p53 and p21. In contrast, the durable G1-arrest by DAP, which failed to activate Chk1 and Chk2, was unaffected by nocodazole.

Conclusions: Cisplatin induced G1-arrest, but at an attenuated level. This was primarily due to orchestration of Cdk inhibition in S-phase first, then in G2, and finally in G1 that effectively blocked cells in G2 and prevented cells from progressing and arresting in G1. These studies demonstrate that cisplatin unequivocally activates G1-checkpoint response, but the fidelity of G1-arrest is compromised by Chk1/2 activation and checkpoint response in S- and G2/M-phase.

Fig. 1

Variation in cell cycle distribution and kinetics with cisplatin. A, A2780 cells were treated with the indicated concentrations of the drug, and analyzed 36 h later. B, Cells were exposed to 1.0 μM cisplatin and analyzed at 6 h intervals for up to 36 h. In each case, analysis was by FACS, with the results presented as percent distribution of cells in different phases of the cell cycle. The data in the bar graphs are shown as mean±SD of 3–4 independent studies.

Fig. 2

Effects of cisplatin on Cdk2/cyclin A and Cdc2/cyclin B kinases. A, A2780 cells were treated with 1.0 μM cisplatin for up to 36 h and lysates prepared at timed intervals. Lysates were immunodepleted with cyclin E antibodies to remove Cdk2/cyclin E complex and then Cdk2 immunoprecipitates isolated and immunoblotted for the indicated proteins and assessed for Cdk2 activity using histone H1 as substrate. B, Cdc2 immunoprecipitates from cells treated with cisplatin as in A were immunoblotted for proteins and assessed for Cdc2 kinase activity using histone H1 as substrate. C, Immunoprecipitates of Cdk2 and Cdc2 isolated from cells treated as in A were immunoblotted with the PY99 antibody to assess tyrosine-15 phosphorylation of Cdk2, or with antibodies that specifically recognize Cdc2 phosphorylated at tyrosine-15. D, Phosphotyrosine-Cdk2 bands in immunoblots from C were quantified by densitometry and plotted against time to show kinetic relationships with Cdk2/cyclin A activity from A and percentage of cells in S-phase from Fig. 1B.

Fig. 3

Effects of cisplatin on the composition, activities and downstream effects of Cdk4/cyclin D1 and Cdk2/cyclin E complexes. A and B, A2780 cells were treated with 1.0 μM cisplatin for up to 36 h and cell lysates from timed samples were immunoprecipitated with either anti-Cdk4 (A) or anti-Cdk2 (B) antibodies. The immunoprecipitates were examined by immunoblot for the indicated proteins and assessed for kinase activity using either Rb (A) or H1 (B) protein as substrate. C, Cells were treated with 1.0 μM cisplatin for up to 36 h and cell lysates from timed samples were immunoblotted for total and phospho-specific Rb antibodies. The total Rb antibody recognizes both hyper- (upper band) and hypophosphorylated (lower band) forms.

Fig. 4

G1-arrest by cisplatin and its dependence on p53 and p21. A and B, A2780 cells were incubated with the indicated concentrations of cisplatin or DAP for 30–36 h and then nocodazole was added 8–10 h later. Cells were collected and the cell cycle distribution quantified by FACS (A) and percentage of cells in different phases of the cell cycle was plotted against cisplatin concentration (B). The data in B are shown as mean±SD of 3–4 independent studies. C, Wild-type, p53−/− and p21−/− HCT-116 cells were treated with cisplatin±nocodazole and processed for cell cycle distribution by FACS as described for A2780 cells in A.

Fig. 5

Differential activation of checkpoint kinase pathways by cisplatin and DAP, and recognition of their DNA adducts by HMG proteins. A, A2780 cells were exposed to 1.0 μM cisplatin or 0.6 μM DAP and harvested at timed intervals over a 30 h period to assess cell lysates for levels of total Chk1, Chk2 and Cdc25A, and phospho-Ser345-Chk1 and phospho-Thr68-Chk2. β-Actin is shown as loading control. B, Cells were transfected with control siRNA oligos or gene-specific duplex siRNA oligos targeting ATR, Chk1, ATM and Chk2 for 5 h and then treated 19 h later with 1.0 μM cisplatin. After a 24-h drug exposure, cell lysates were prepared and immunoblotted to examine the specified proteins. Note that in the ATR immunoblot, the upper band is ATR and the lower bands are non-specific. C, Experiments in B were repeated, but using 0.6 μM DAP. D, Nuclear proteins binding to DNA-cellulose adducts of cisplatin or DAP were separated by SDS-PAGE and silver-stained to visualize HMGB1 and HMGB2 proteins. Lane 1, nuclear proteins bound to 40 μg control (cont) untreated DNA-cellulose; lanes 2–7, nuclear proteins bound to 4–40 μg of DNA-cellulose following treatment with either cisplatin (lanes 2–4) or DAP (lanes 5–7); lane 8, total nuclear proteins (prot).

Fig. 6

A proposed model for primary cell cycle effects of cisplatin and DAP. Based on their chemical structures, cisplatin and DAP form qualitatively different intrastrand adducts, which are shown in the Pt(II) form although the Pt(IV) state for the DAP adduct is possible [50]. The damaged DNA after cisplatin and DAP is differentially recognized by independent proteins to activate downstream kinases that subsequently induce p53, which then transactivates p21 to inhibit G1-phase Cdk. In addition, the ATR, Chk1 and/or Chk2 activated by only cisplatin downregulate Cdc25, which then inhibits both Cdk2 in S-phase and Cdc2 in G2/M by increasing phosphorylation at Y-15. The approximate times at which cell cycle arrest is observed in each of the specific phases are shown to explain how the sequence of S- and G2/M-arrests by cisplatin obscures G1-arrest.