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KMID : 0366220020370030200
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Korean Journal of Hematology
2002 Volume.37 No. 3 p.200 ~ p.211
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Arsenic Trioxide (As2O3) Induced Apoptosis in NB4 Cell lines
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Park Kyeong-Bae
Hong Dae-Sik
Suh Won-Suk
Lee Nam-Su
Park Sung-Kyu
Won Jong-Ho
Jung Hee-Jung
Kim Sook-Ja
Hong Young-Seon
Jin Jong-Youl
Park Hee-Sook
Shin Sang-Mann
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Abstract
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Background: Recently, inorganic arsenic trioxide (As2O3) was reported to induce complete remission in high proportion of patients with refractory promyelocytic leukemia (APL). To illustrate cellular and molecular me chanisms of As2O3 in the treatment of APL, many experimental studies were performed on APL-derved cell lines in vitro. Previous studies showed that As2O3 inhibited proliferation and induced apoptosis in the APL-derved cell lines. This study was done to clarify the invitro mechanisms of As2O3-induced apoptosis in APL-derived NB4 cell lines.
Method: To determine the effects of As2O3 in the various concentrations, NB4 cells were cultured with 0.1 to 2μM/L of As2O3. To assay the apoptosis in NB4 cell lines, DNA fragmentation assay and TUNEL were performed. To find out the molecular change of As2O3-induced apoptotic NB4 cell lines, RT-PCR and Western blot analysis for PML-RARα chimeric protein expression and flow cytometry for bcl-2/bax expression were performed. To clarify the caspase activation pathway, Western blot analysis and flow cytometry for procaspase expression were performed.
Results: As2O3 induces apoptosis on NB4 cells in relatively high concentration (0.5 to 2μM/L) for 2 days. After 2 days of culture the PML-RARα chimeric protein expression decreased rapidly by Western blot and RT-PCR analysis and bcl-2 expression also decreased by flow cytometry. The expression of bax by flow cytometry showed a marked increase in high concentration (2μM/L) but there was no change in low concentration (0.5μM/L). In the Western blot analysis, the amount of proenzyme of caspase-3 was significantly decreased in the cells with high concentration (2μM/L) compared with that in the cells with low concentration (0.5μM/L). As2O3 induces proteolytic processing of pro-caspase 7 but not pro-caspase 9 and 8.
Conclusion: Apoptosis of APL-derved NB4 cell lines was induced by As2O3 and progressed rapidly in higher concentrations. During aoptosis, activation of caspase-7 pathway and degradation of PML-RARα chimeric protein, decrease in bcl-2 and increase in bax were shown.
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KEYWORD
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Arsenic trioxide (As2O3) Apoptosis, NB4 Cell lines, Caspase
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