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KMID : 0357119990210010025
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Korean Journal of Immunology
1999 Volume.21 No. 1 p.25 ~ p.34
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A Study on Apoptosis of HGPRT- Mouse Myeloma Cell induced by Aminopterin, a de novo Pathway Blocking Agent
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Yong Choi
Yong-Hoon Chung
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Abstract
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Programmed cell death (PCD), or apoptosis, plays an important role in embryonic development, metamorphsis, hormone-dependent atrophy and tumor growth, as a physiological event regulating the cell number or eliminating damaged cells. Currently hybridoma cell production, resulting from the fusion of myeloma cells with antibody producing spleen cells, is widely used in various fields of life science. This technique requires a hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficient mutant myeloma cell line as a fusion partner. When these rnutant cells are treated with aminopterin plus hypoxanthine-thymidine (HAT) after the cell fusion they are selectively and efficiently eliminated so that one could get only fused hybridoma cells. But there hasn't been any report regarding this selective elimination mechanism of HGPRT mutant myeloma cell. By using HGPRT myeloma V653 as a model system this study demonstrated that PCD was induced by aminopterin treatment of this V653 cell. And this PCD was further characterized by cotreatement of cycloheximide, actinomycin-D, and calcium ionophore A23187 together with aminopterin. The apoptotic endonuclease involved in this PCD process was also detected and characterized. When V653 cells were incubated for the various periods of time in the presence of 0.4 uM aminopterin, the viability was continued to decrease until 48 hours of aminopterin treatment and there was no viable cell affer 36 hours of incubation. DNA fragmentation was detectable 3 hours of incubation and peaked between 12 and 18 hours of aminopterin treatment. The induction of cell death and DNA fragmentation of V653 cells by aminopterin were inhibited by protein synthesis inhibitor, cycloheximide, and RNA synthesis inhibitor, actinomycin-D and maximal inhibitory effects on cell death were seen at concentrations of 2 ug/ml and 0.5 uM, respectively. Ca2+ ionophore A23187 promoted aminopterin-induced cell death of V653. When the cells were coincubated with A23187 in the presence of aminopterin, cell viability was remarkably decreased at concentrations of more than 2 uM and DNA fragmentation was increased at concentrations of more than 0.2 uM. A23187 also induced cell death when the cells were treated with A23187 alone. When endogenous endonuclease activities of nuclei isolated from intact healthy cells and aminopterin-treated cells were compared for four different conditions, there were notable increases in the Ca2+/Mg2+ -independent and the Mg2+ -dependent endonuclease activity after incubation with aminopterin for 12 hours. In northern blot analysis, induction of c-myc gene was observed in aminopterin-treated V653 cell reached peak at 2 hours and thern decreased drastically.
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KEYWORD
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Apoptosis, Programmed cell death, Myeloma cell,
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