[细胞色素P450 IIIA亚家族多肽5基因与白血病细胞系耐药性的关系]

[The relationship between cytochrome P450, subfamily IIIA, polypeptide 5 gene and drug resistance in leukemia cell lines].

作者信息

Wang Ting, Chen Fang-yuan, Han Jie-ying, Zhong Ji-hua, Teng Ye, Ouyang Ren-rong

机构信息

Department of Hematology, Renji Hospital of Shanghai Second Medical University, Shanghai Institute of Hematology, Shanghai 200001, China.

出版信息

Zhonghua Nei Ke Za Zhi. 2004 Jul;43(7):527-9.

PMID:15312409

Abstract

OBJECTIVE

To investigate if the transcription, expression and activities of cytochrome P450, subfamily IIIA, polypeptide 5 gene (CYP3A5) are correlated with drug resistance in leukemia cell lines.

METHODS

Using RT-PCR, immunohistochemistry and reverse-phase high-performance liquid chromatography (HPLC) assay, CYP3A5 mRNA protein and activities in leukemia cell lines were detected. Daunorubicin (DNR) sensitivity profiles of leukemia cell lines were obtained with MTT assay. Cell cycle characteristics and apoptosis induced by DNR were observed with flow cytometry (FCM) analysis.

RESULTS

K562, U937, HL-60, NB4 and Jurkat cells were chosen as experimental cell line panels. It was found that CYP3A5 mRNA were measured only in K562 and U937 cells. CYP3A4 and CYP3A7 were not detectable in each cell line. The five leukemia cell lines presented sensitivity to DNR (IC(50), mg/L) in an order as follows: NB4 (0.068 +/- 0.036) > Jurkat (0.076 +/- 0.013) > HL-60 (0.092 +/- 0.016) > K562 (0.148 +/- 0.041) > U937 (0.150 +/- 0.035). Interestingly, K562 and U937 cells (cell lines with CYP3A5 gene transcription) were more resistant to DNR as compared with the other three cell lines in a statistically significant way (P < 0.01). Furthermore, expression and activities of CYP3A5 gene and cell cycle characteristics were observed in K562 and NB4 cells, which represented cell lines with or without CYP3A5 gene transcription respectively. In comparison with NB4 cells, K562 cells expressed CYP3A5 protein and showed a statistically significant higher CYP3A5 activity (3.075 +/- 0.036) x 10(-3) versus (1.635 +/- 0.196) x 10(-3), P < 0.05. FCM analysis showed that S phase cell proportions were similar in K562 and NB4 cells. Incubation with DNR (1 mg/L) for 6 hours induced a remarkable apoptosis peak in NB4 cells, while such peak not occur in K562 cells (apoptosis cell percentage 18.4% and 0.8% respectively).

CONCLUSION

Only CYP3A5 gene of CYP3A subfamily were detectable in leukemia cell lines, and its transcription, expression and activities were correlated with drug resistance in leukemia cell lines.

摘要

目的

研究细胞色素P450 3A亚家族多肽5基因（CYP3A5）的转录、表达及活性是否与白血病细胞系的耐药性相关。

方法

采用逆转录聚合酶链反应（RT-PCR）、免疫组织化学和反相高效液相色谱（HPLC）分析法，检测白血病细胞系中CYP3A5 mRNA、蛋白及活性。用MTT法检测白血病细胞系对柔红霉素（DNR）的敏感性。通过流式细胞术（FCM）分析观察DNR诱导的细胞周期特征和凋亡情况。

结果

选择K562、U937、HL-60、NB4和Jurkat细胞作为实验细胞系。发现仅在K562和U937细胞中检测到CYP3A5 mRNA。在各细胞系中均未检测到CYP3A4和CYP3A7。五种白血病细胞系对DNR的敏感性（IC(50)，mg/L）顺序如下：NB4（0.068±0.036）>Jurkat（0.076±0.013）>HL-60（0.092±0.016）>K562（0.148±0.041）>U937（0.150±0.035）。有趣的是，与其他三种细胞系相比，K562和U937细胞（具有CYP3A5基因转录的细胞系）对DNR的耐药性在统计学上有显著差异（P<0.01）。此外，分别观察了代表有或无CYP3A5基因转录的细胞系K562和NB4细胞中CYP3A5基因的表达、活性及细胞周期特征。与NB4细胞相比，K562细胞表达CYP3A5蛋白，且CYP3A5活性在统计学上显著更高（(3.075±0.036)×10(-3)对(1.635±0.196)×10(-3)，P<0.05）。FCM分析显示，K562和NB4细胞中S期细胞比例相似。用1mg/L DNR孵育6小时后，NB4细胞诱导出明显的凋亡峰，而K562细胞未出现此峰（凋亡细胞百分比分别为18.4%和0.8%）。