Korean J Hematol 2002; 37(3):
Published online September 30, 2002
© The Korean Society of Hematology
As2O3에 의한 NB4 백혈병 세포주에서 아포프토시스의 유도
박경배, 홍대식, 서원석, 이남수, 박성규, 원종호, 정희정, 김숙자, 홍영선, 진종률, 박희숙, 신상만
순천향대학교 의과대학 소아과학교실,
임상분자생물학연구소,
순천향대학교 의과대학 내과학교실,
가톨릭대학교 의과대학 내과학교실
Arsenic Trioxide (As2O3) Induced Apoptosis in NB4 Cell lines
Background : Recently, inorganic arsenic trioxide (As 2 O 3 ) was reported to induce complete remission in high proportion of patients with refractory promyelocytic leukemia (APL). To illustrate cellular and molecular me chanisms of As 2 O 3 in the treatment of APL, many experimental studies were performed on APL-derved cell lines in vitro. Previous studies showed that As 2 O 3 inhibited proliferation and induced apoptosis in the APL-derved cell lines. This study was done to clarify the invitro mechanisms of As 2 O 3 -induced apoptosis in APL-derived NB4 cell lines.
Method: To determine the effects of As 2 O 3 in the various concentrations, NB4 cells were cultured with 0.1 to 2μM/L of As 2 O 3 . To assay the apoptosis in NB4 cell lines, DNA fragmentation assay and TUNEL were performed. To find out the molecular change of As 2 O 3 -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). As2 O 3 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 As 2 O 3 andprogressed 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.
Keywords Arsenic trioxide (As2O3); Apoptosis; NB4 Cell lines; Caspase;
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Korean J Hematol 2002; 37(3): 200-211
Published online September 30, 2002
Copyright © The Korean Society of Hematology.
As2O3에 의한 NB4 백혈병 세포주에서 아포프토시스의 유도
박경배, 홍대식, 서원석, 이남수, 박성규, 원종호, 정희정, 김숙자, 홍영선, 진종률, 박희숙, 신상만
순천향대학교 의과대학 소아과학교실,
임상분자생물학연구소,
순천향대학교 의과대학 내과학교실,
가톨릭대학교 의과대학 내과학교실
Arsenic Trioxide (As2O3) Induced Apoptosis in NB4 Cell lines
Kyeong Bae Park, Dae Sik Hong, Won Suk Suh, Nam Soo Lee, Sung Gyu Park, Jong Ho Won, Hee Jung Jung, Sook Ja Kim, Young Seon Hong, Jong Youl Jin, Hee Sook Park, Sang Man Shin
Department of Pediatrics and Internal Medicine and Institute for Clinical Molecular Biology Research, College of Medicine, Soon Chun Hyang University
Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea
Abstract
Background : Recently, inorganic arsenic trioxide (As 2 O 3 ) was reported to induce complete remission in high proportion of patients with refractory promyelocytic leukemia (APL). To illustrate cellular and molecular me chanisms of As 2 O 3 in the treatment of APL, many experimental studies were performed on APL-derved cell lines in vitro. Previous studies showed that As 2 O 3 inhibited proliferation and induced apoptosis in the APL-derved cell lines. This study was done to clarify the invitro mechanisms of As 2 O 3 -induced apoptosis in APL-derived NB4 cell lines.
Method: To determine the effects of As 2 O 3 in the various concentrations, NB4 cells were cultured with 0.1 to 2μM/L of As 2 O 3 . To assay the apoptosis in NB4 cell lines, DNA fragmentation assay and TUNEL were performed. To find out the molecular change of As 2 O 3 -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). As2 O 3 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 As 2 O 3 andprogressed 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.
Keywords: Arsenic trioxide (As2O3), Apoptosis, NB4 Cell lines, Caspase,
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