Elsevier

Gynecologic Oncology

Volume 122, Issue 2, August 2011, Pages 402-409
Gynecologic Oncology

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

https://doi.org/10.1016/j.ygyno.2011.04.034Get rights and content

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-116p53−/− and HCT-116p21−/− 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.

Research highlights

► Cisplatin potently inhibits G1-phase Cdk in a p53- and p21-dependent manner. ► Activation of Chk1 and Chk2 inhibits Cdk in S- and G2-phase and impedes G1-arrest. ► ATM, ATR, Chk1 and Chk2 kinases are redundant for activity of a Pt(IV) analog DAP.

Introduction

Cisplatin is highly effective in the treatment of ovarian cancer. Its mechanism of action is ascribed to intrastrand binding to DNA, with potent cellular signaling events that eventually lead to apoptosis. A general critical event associated with DNA damage is activation of cell cycle checkpoints, which ultimately result in inhibition of cyclin-dependent kinase (Cdk) complexes. Upstream events in checkpoint response involve activation of ATR and ATM kinases, with resultant increase in Chk1 and/or Chk2 phosphorylation to regulate cell cycle progression [1], [2]. More specifically, S- and G2-phase checkpoint responses are manifested by Chk1/2-dependent downregulation of Cdc25 phosphatase, with the consequence that Cdk within the Cdk2/cyclin A and Cdc2/cyclin B complexes remain in the inhibitory tyrosine phosphorylated state. Although all three isoforms of Cdc25 (Cdc25A, Cdc25B and Cdc25C) may dephosphorylate Cdk2 and Cdc2, recent knock-out studies indicate that Cdc25A is the more critical [3]. G1-checkpoint response is also dependent on upstream kinases, which stabilize p53 to transactivate p21 and inhibit G1-phase Cdk4/cyclin D and Cdk2/cyclin E complexes [1], [2].

Cdk inhibition in G1, S and/or G2 phases normally leads to cell cycle retardation or arrest, which allows DNA repair and prevents DNA replication or mitosis in the presence of genomic damage. When repair fails, DNA damage leads to apoptosis. Therefore, cell cycle arrest and cell fate are intimately linked, and understanding their inter-relationship has the potential for clinical benefits [1], [4], [5]. In this regard, platinum-based agents demonstrate potent activity only against tumor cells proficient in G1-arrest [6], which is consistent with the development of cisplatin resistance when p21 cannot be upregulated [7], [8]. Since the experimental non-cross-resistant analog 1R,2R-diaminocyclohexane(trans-diacetato)(dichloro)platinum(IV) (DAP) is a potent inducer of G1-arrest, and largely devoid of S- and G2/M-arrest [9], it supports the notion that activation of G1-checkpoint response is important for platinum-mediated antitumor effects.

Although G1-checkpoint response or arrest correlates with platinum drug activity, it is paradoxical that cisplatin is not associated with G1-arrest; instead, it predominantly induces a transient S-phase arrest that is followed by a robust G2/M-arrest, irrespective of the p53 status [7], [10], [11], [12], [13], [14]. Indeed, there has been no definitive study to examine the effect of cisplatin on G1-phase Cdk complexes, particularly in parallel with S- and G2/M-checkpoint responses. Where G1-arrest by cisplatin has been reported, these are largely observed in p53-defective cells (e.g., HeLa cells [15]) and/or at high cisplatin concentrations (e.g., > 5 μM [13], [15]), the mechanism for which is not understood. Therefore, to examine the potential of G1-checkpoint response with cisplatin, we have undertaken a systematic biochemical and molecular analysis in ovarian p53-proficient A2780 cells, and used DAP as a positive control for G1-arrest and a negative control for S- and G2/M-arrest in this tumor model system [9], [16].

Section snippets

Cell culture and drug treatment

A2780, wild-type HCT-116wt, p53-deficient HCT-116p53−/− and p21-deficient HCT-116p21−/− cells were maintained as previously described [16]. Cells were plated in 100 mm dishes and incubated for at least 24 h before being exposed to freshly prepared cisplatin or DAP. The cells were further cultured and collected at selected times. Drug-treated cultures also received nocodazole, where required. Fluorescence activated cell sorting (FACS) and extraction of total proteins for biochemical assays were

Dose-response and temporal effects of cisplatin on cell cycle progression

A2780 cells exposed to cisplatin accumulated in G2/M, and this was initially seen at 1.0 μM drug concentration, and becoming maximal at 2.0 μM (Fig. 1A). Concomitant decreases in S- and G1-phase populations were observed, but higher concentrations increased relative distribution of cells in S-phase and decreased it in G2/M. G1 Accumulation was not observed at any concentration. Based on these data, a cisplatin concentration of 1.0 μM (~ 5 × IC50 in this model [24]) was selected for all remaining

Discussion

Cell cycle arrest in a given phase of the cell cycle is indicated when cell numbers in that phase increase above control levels. Based on this limited criterion, G1-arrest is not observed with cisplatin. However, cisplatin in our detailed investigation induced not only the expected S- and G2/M-arrests, but also G1-arrest, which was consistent with inhibition of G1-phase Cdk4/cyclin D1 and Cdk2/cyclin E complexes and the dependence on p53 and p21. The low level of G1-arrest by cisplatin was due

Conflict of interest statement

The authors have no conflict of interest to declare.

Acknowledgment

We are thankful to Mr. Long Nguyen for technical assistance in the HMGB studies.

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    Grant support: U.S. Public Health Service grants CA127263 to Z.H.S. and CA16672 to MD Anderson Cancer Center, awarded by the National Cancer Institute, and in part to the Megan McBride Franz Endowed Research Fund.

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