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☆
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.
References (50)
- et al.
Characterization of high mobility group protein binding to cisplatin-damaged DNA
Biochem Biophys Res Commun
(1992) - et al.
Purification of nuclear proteins that bind to cisplatin-damaged DNA. Identity with high mobility group proteins 1 and 2
J Biol Chem
(1992) - et al.
A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing
Gene Anal Tech
(1988) - et al.
Role of p53 in the ability of 1,2-diaminocyclohexane-diacetato-dichloro-Pt(IV) to circumvent cisplatin resistance
J Inorg Biochem
(1999) - et al.
Differential impact of diverse anticancer chemotherapeutics on the Cdc25A-degradation checkpoint pathway
Exp Cell Res
(2005) - et al.
Cisplatinum and taxol induce different patterns of p53 phosphorylation
Neoplasia
(2001) - et al.
p21/CDKN1A mediates negative regulation of transcription by p53
J Biol Chem
(2003) - et al.
The cell cycle inhibitor p21waf1 binds to the myc and cdc25A promoters upon DNA damage and induces transcriptional repression
J Biol Chem
(2006) Cell cycle checkpoints and their impact on anticancer therapeutic strategies
J Cell Biochem
(2004)- et al.
Linking DNA damage to cell cycle checkpoints
Cell Cycle
(2002)