Original contributionRole of reactive oxygen species and MAPKs in vanadate-induced G2/M phase arrest
Introduction
Vanadium is an essential transition trace element found in some plants and animals. It is widely distributed in rocks, soil, and to a lesser extent in water 1, 2, 3. Vanadium compounds (V(V)) exert potent toxic effects on a wide variety of biological systems 1, 4, 5, 6, 7, 8. This metal regulates growth factor-mediated signal transduction pathways, promotes cell transformation, and decreases cell adhesion 9, 10, 11. Occupational exposure to vanadium occurs in mining, petrochemical industries, and coal- and oil-fired plants. Epidemiological studies have shown a correlation between vanadium exposure and the incidence of lung cancer in humans 6, 8, 12, 13. Vanadium compounds were reported to modify DNA synthesis and repair 14, 15, 16. Vanadate induced forward mutations and DNA-protein cross-links in cultured mammalian cells [17]. While the biochemical mechanism of vanadium carcinogenicity still is not fully understood, recent studies have indicated that vanadium-mediated generation of reactive oxygen species (ROS) may play an important role 18, 19, 20, 21, 22, 23, 24, 25, 26. For example, through ROS, vanadium caused 2′-deoxyguanosine hydroxylation and DNA damage [26], apoptosis 27, 28, and activation of nuclear transcription factors [21] AP-1 and NF-κB [29].
In mammalian cells, cell cycle transition is under the control of a tightly regulated network of cell division kinases (cdks) and numerous surveillance mechanisms, the so-called checkpoints [30]. In most normal cells, DNA damage arrests proliferation in G1/S or G2/M phase and then resumes proliferation after the damage is repaired [30]. The cell cycle controls the onset of DNA replication and mitosis to ensure the integrity of the genome 31, 32, 33. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations, leading to cell death or, in multicellular organisms, cancer [30].
Recent evidence indicates that ROS may function as intracellular messengers to modulate signaling pathways 34, 35. The changes of intracellular ROS have been detected in a variety of cells stimulated with cytokines, growth factors, and agonists of receptors 34, 36, 37. Various experiments have shown that many protein kinases and transcription regulatory factors are activated under the conditions of oxidative stress 38, 39, 40, 41, 42, 43. Mitogen-activated protein kinases (MAPKs) cascades are protein kinase signal transduction pathways that have been remarkably conserved in evolution. They are differentially used to relay numerous extracellular signals within cells 44, 45, 46. These MAPK cascades have been found to be involved in such diverse cellular functions as proliferation, differentiation, stress responses, and apoptosis.
Stress-activated protein kinases (SAPK)/Jun N-terminal kinase (JNK), p38, and extracellular signal-related kinase (ERK) are the most widely studied members of the MAPK family. Activation of MAPKs led to abnormal M phase transition in the cell cycle [47]. It has been reported that p38 functions as a component of the spindle assembly checkpoint in somatic cell cycles [48]. Although ROS are frequently mentioned in the literature to be inducers for MAPKs, many of the studies are indirect. For example, N-acetylcysteine (NAC) was used as an inhibitor [49] and the generation of ROS by the stimulant in cellular system was not well characterized. NAC can readily react with “–SH” group of the protein and affect its function in a mechanism other than the scavenging of ROS.
The link between ROS and cell growth arrest has not yet been established. Many questions remain to be answered. For example, do ROS play a key role in the induction of cell growth arrest? Which species among ROS are involved? Are MAPKs involved in ROS-mediated cell growth arrest? Do MAPKs and ROS affect cell growth regulatory proteins? The present study attempts to answer these questions using vanadate as an inducer.
Section snippets
Chemicals
Sodium metavanadate was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). RNase A and superoxide dismutase (SOD) were from Sigma Chemical Co. (St. Louis, MO, USA). Catalase was from Roche Molecular Biochemicals (Indianapolis, IN, USA). Propidium iodide (PI), 2′, 7′-dichlorofluorescin diacetate (DCFH-DA), and dihydroethidium (HE) were from Molecular Probes (Eugene, OR, USA). Both F12K nutrient mixture medium and fetal bovine serum (FBS) were from Gibco BRL (Life Technologies,
The effect of vanadate on the cell cycle
DNA content was measured by flow cytometry to investigate vanadate-induced cell growth arrest. Human alveolar epithelial cells (the A549 cell line) were first synchronized by serum starvation and then exposed to vanadate for 24 h with various concentrations of vanadate. The results show that exposure of the cells to 100 μM vanadate caused growth arrest at the G2/M phase in agreement with a previous report [55]. At a vanadate concentration of 100 μM, vanadate-induced G2/M phase arrest peaked
Discussion
Although vanadate-containing compounds exert potent toxic and carcinogenic effects on a wide variety of biological systems, the mechanisms of their actions remain to be investigated. We hypothesize that vanadate-mediated free radical reactions may cause oxidative stress and play a key role in the mechanism of vanadate-induced carcinogenesis 24, 25. ROS generated by vanadate-mediated reactions are able to damage DNA [26]. Normally, if the cell is injured by external agents, it will respond to
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