Mechanism underlying cytotoxicity of thialysine, lysine analog, toward human acute leukemia Jurkat T cells
Introduction
Thialysine, S-2-aminoethylcysteine, is a lysine analog with the 4-methylene group substituted by a sulfur atom. Since the structural similarity of thialysine to lysine is remarkable, it can effectively compete with lysine for lysyl tRNA activation and for incorporation into cellular proteins. Previously it has been shown that thialysine is toxic against microorganisms including bacteria [1], [2] and yeasts [3], [4], and reduces the growth rate and plating efficiency of Chinese Hamster Ovary (CHO) cells [5]. These toxic effects of thialysine appear to be reversible by the addition of lysine. Several studies have proposed the possible application of amino acid analogs to pharmacological treatment of malignant conditions including cancers [6], [7], [8], [9], [10], [11], [12]. For instance, the l-arginine analog, l-canavanine has been reported to possess growth retardation activity toward tumor cells in culture and experimental tumors in vivo[6], [7], [11]. Synergic antitumor effects from a combination of l-canavanine with 5-fluorouracil or γ-irradiation have been demonstrated, indicating that l-canavanine may modulate the chemo- or radio-sensitivity of tumors [8], [9]. In addition, it has been shown that the amino acid analog, l-2,4 diaminobutyric acid accumulates in hepatoma cells, which causes hyperosmosis with subsequent cell lysis [12]. Although these suggest that the inhibitory mode of amino acid analogs toward tumors may vary depending on the analog types and cell types, a primary mechanism underlying the antitumor activity of amino acid analogs, leading to cell damage, is thought to be their incorporation into cellular proteins in substitution for the intact forms and subsequent induction of structurally aberrant proteins with impaired function or degradation [13], [14], [15]. Chemotherapy employing antineoplastic drugs often relies on the difference of the mitotic rate between tumor and normal cells in order to confine its toxic effect to the tumor. In this regard, amino acid analogs have been simply considered to possess a potency as chemotherapeutic agents because their incorporation into cellular proteins, which result in an inhibitory effect on cell growth, can be more significant in tumor cells than in normal cells. However, the inhibitory activity of amino acid analogs on the growth of tumor cells, and the underlying inhibitory mechanisms requisite for evaluating their potency as a chemotherapeutic agent, remain largely unknown. Since the induction of apoptosis in tumor cells can lead to their own destruction, apoptosis has been implicated as an efficient mechanism by which malignant tumor cells are removed when treated with antineoplastic drugs. As a potential mechanism in the drug-induced apoptosis, upregulation of Fas ligand (FasL) and/or Fas expression with subsequent induction of apoptotic cell death through activation of Fas signaling has been implicated [16], [17], [18], [19]. Mitochondrial release of cytochrome c-mediated activation of caspase–cascade has also been implicated in chemotherapeutic agent-induced apoptosis [20], [21]. However, the involvement of apoptotic cell death in the inhibitory activity of amino acid analogs against tumor cells is still poorly elucidated.
In a previous study, we found that the cytotoxicity of l-canavanine toward human acute leukemia Jurkat T cells is attributable to induced apoptosis [22]. l-Canavanine-induced apoptosis appeared to accompany mitochondrial cytochrome c-independent activation of caspase-3, which could be interrupted by an ectopic expression of Bcl-2 or Bcl-xL, suggesting that l-canavanine may cause apoptotic cell death of Jurkat T cells by triggering a conserved caspase cascade, leading to caspase-3 activation without involving the mitochondrial cytochrome c release. Since mitochondria are known to play an important role in the commitment of apoptosis provoked by many physiological and non-physiological signals [23], [24], [25], our previous results raised a question that the mitochondria-independent pathway converging to caspase-3 activation plays a central role in amino acid analog-induced apoptosis regardless of the analog types. In the present study, to understand the mechanism by which amino acid analogs induce apoptotic cell death, we investigated a lysine analog, thialysine-induced apoptotic signaling pathway in Jurkat T cells, focusing on the mitochondrial cytochrome c-mediated activation of caspase cascade. In addition, we compared a thialysine-mediated alteration in the cell cycle distribution of Jurkat T cells transfected with Bcl-xL gene (J/Bcl-xL) and Jurkat T cells transfected with vector (J/Neo) to investigate whether thialysine arrests cell cycle progression. The results demonstrate that thialysine induces apoptotic cell death in Jurkat T cells via a mitochondria-dependent death signaling pathway including mitochondrial cytochrome c release and activation of caspase-9 and -3, which is negatively regulated by Bcl-xL. The results also demonstrate that thialysine can arrest cell cycle progression by inducing down-regulation of the protein levels of several positive cell cycle regulators, indicating that cytotoxic activity of thialysine toward malignant Jukart T cells is due to induced apoptosis as well as the cell-cycle arrest.
Section snippets
Reagents, antibodies, and cells
Thialysine and decylubiquinone were purchased from Sigma Chemical. The ECL Western blotting kit was from Amersham. Anti-cytochrome c antibody was purchased from Pharmingen. Anti-caspase-9, anti-caspase-3, anti-PARP, and anti-Bcl-xL were from Santa Cruz Biotechnology. Monoclonal anti-FasL antibody was purchased from Transduction Laboratories. A broad-spectrum caspase inhibitor (z-VAD-fmk) was purchased from Calbiochem. Human acute leukemia Jurkat T cell line E6.1 was supplied by Dr. Albert A.
Apoptotic effect of thialysine on Jurkat T cells
To understand the mechanisms underlying the cytotoxicity of thialysine, its effect on Jurkat T cell line E6.1 was investigated. When Jurkat T cells were treated with thialysine at various concentrations ranging from 0.32 to 2.5 mM for 20 hr, cell viability determined by MTT assay appeared to decline significantly in a dose-dependent manner (Fig. 1A). After treatment with 0.32 mM thialysine, the viability remained at the level of 40%. Cell viability declined, however, to a minimal level (20%) in
Acknowledgements
This work was supported by the Korean Research Foundation Grant (KRF-2000-015-DP0310). Dr. D.Y. Jun is the recipient of a Post-Doctoral Fellowship from Kyungpook National University, Taegu, Korea (2001–2002).
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