Genistein arrests hepatoma cells at G2/M phase: involvement of ATM activation and upregulation of p21waf1/cip1 and Wee1
Introduction
The phytoestrogen isoflavones present in soybeans have a wide range of properties that may contribute to their cancer chemoprotective actions [1]; these include an inhibitory effect on tyrosine kinases, DNA topoisomerases I and II, and ribosomal S6 kinase, anti-estrogenicity, antioxidant activity, anti-angiogenesis activity, suppression of cell proliferation, induction of differentiation, and modulation of apoptosis [2], [3], [4], [5]. In addition, genistein (5,7,4′-trihydroxyisoflavone), one of the most active of these compounds, is an effective chemopreventive agent in dimethylbenz[α]anthracene-induced mammary carcinogenesis in neonatal rats [6]. Our previous study [7] showed that genistein has an antiproliferative effect on human hepatoma cancer cell lines, as previously reported for a number of other cell types [8], [9]. However, its mechanism of action and its molecular targets on human hepatoma cells remain unclear.
Cell cycle checkpoints are biochemical pathways that ensure the orderly and timely progression and completion of critical events, such as DNA replication and chromosome segregation. The ultimate target of the G2 checkpoint signaling pathway is the cyclin-dependent kinase (Cdk) complex, Cdk1-cyclin B1. Cdc2, a Cdk1 first discovered in Schizosaccharomyces pombe, forms a heterodimeric complex with cyclin B1 which is maintained in an inactive form by phosphorylation of residues Thr-14 and Tyr-15 in the ATP-binding domain of Cdc2 by Wee1 kinase [10], [11] and is converted to an active form by dephosphorylation of these residues by the dual specificity phosphatase, Cdc25C [12]. This dephosphorylation/activation is an absolute requirement for the onset of mitosis. Recently, it has been shown that Cdc25C is negatively regulated by phosphorylation of its Ser-216 residue during interphase or in response to DNA damage or incomplete DNA replication [13]. Phosphorylation of this residue creates a binding site for 14-3-3 proteins, which are believed to be responsible for the nuclear export of Cdc25C and the subsequent inhibition of nuclear Cdk1 dephosphorylation. Two checkpoint kinases, Chk1 and Chk2, have been recently identified in humans and shown to phosphorylate Cdc25C on Ser-216 [13], [14]. Chk1 is activated in γ-irradiated HeLa cells [14], whereas Chk2 is involved in both the replication and DNA damage checkpoints [15]. Although the initial study [14] demonstrated that Chk1 could be activated by ionizing radiation, more recent studies clearly showed that this activation is only observed at high doses and that Chk2 is markedly more responsive to ionizing radiation than Chk1 [15], [16]. The response to DNA damage is dependent on the ATM protein signaling pathway [17]. ATM, a member of the phosphatidylinositol 3-kinase (PI 3-K) protein family [18], is required for the activation of p53, a tumor suppressor protein, and of Chk2, which results, respectively, in cell cycle arrest at G1/S or G2/M [19].
A 21-kD protein, p21waf1/cip1, is a component of Cdk-cyclin complexes and can modulate the activity of a number of Cdks [20]. Association of p21waf1/cip1 with Cdk-cyclin complexes results in decreased Cdk activity, which, in turn, inhibits the phosphorylation/inactivation of specific endogenous substrates, such as the retinoblastoma protein, which are required for progression through the cell cycle [21]. Additionally, p21waf1/cip1 is a universal Cdk inhibitor and causes cell cycle arrest at G1/S or G2/M [20], [22].
We have recently reported that genistein arrests hepatoma cells at G2/M phase and inhibits Cdc2 kinase activity [7]. The aims of the present study were, therefore, to examine the effectiveness of genistein in modulating Wee1 and p21waf1/cip1 expression and Cdc25C phosphorylation status, and to address the question of whether genistein exerts these effects by activating an ATM kinase and, if so, whether this activation involves p53 or Chk2.
Section snippets
Reagents
Genistein, DMSO, and protein A-Sepharose were purchased from Sigma Chemical Co. Horseradish peroxidase-conjugated secondary antibodies and antibodies against p53, p21waf1/cip1, Chk2, Cdc25C, Cdc25C-P, Cdc2, Cdc2-P, cyclin B1, and β-actin were purchased from Santa Cruz Biotechnology Inc. Anti-ATM antibody and PHAS-1 were purchased from Oncogene Research Products. Histone H1 was purchased from Calbiochem-Novabiochem. Cell growth media and serum were obtained from GIBCO BRL. []ATP (3000
Genistein arrest HepG2 cells at G2/M phase
To gain an initial insight into the effects of genistein on cell cycle distribution, the human hepatoma cell line, HepG2, was incubated for 24 hr with 0–30 μg/mL genistein. As shown in Fig. 1, increasing concentrations of genistein caused increasing accumulation of cells in G2/M phase.
Effects on cyclin B1 expression and Cdc2 activity and phosphorylation
To examine the expression of intracellular proteins regulating cell cycle progression at the G2/M boundary, cyclin B1 expression and Cdc2 kinase activity were analyzed. When cells were incubated for 24 hr with 0–20
Discussion
In this study, we found that genistein treatment decreased Cdc2 activity, increased Cdc2 phosphorylation, and enhanced the expression of the cell cycle inhibitor, p21waf1/cip1. Furthermore, it increased both the levels of phosphorylated/inactivated Cdc25C phosphatase and Wee1 kinase. From these results, we conclude that genistein arrests HepG2 cells at G2/M phase (i) by inhibiting Cdc2 dephosphorylation/activation by causing a decrease in Cdc25C levels and an increase in Wee1 levels, and (ii)
Acknowledgements
This study was supported by the National Science Council, Executive Yuan, Taiwan (Grant NSC-90-2320-B-242-003 and NSC 91-2320-B037-035).
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