急性白血病凋亡因子及多药耐药的实验和临床研究
摘要
目前化学治疗仍是临床治疗白血病的主要手段，也是造血干细胞移植的基础。近年来，尽管有多种新的药物和治疗方案推出，但大多数急性白血病(AL)患者最终因治疗失败而死亡，导致治疗失败的原因是多方面的，其中多药耐药(MDR)是最重要的一种。多种机制可导致MDR，涉及P-糖蛋白(P-gp)和多药耐药相关蛋白(MRP)的药物泵出、肺耐药蛋白(LRP)和乳腺癌耐药蛋白(BCRP)的药物转运、细胞凋亡受抑以及酶解毒增强等。为较全面地了解AL患者体内的耐药蛋白和凋亡调节蛋白的表达、及其与临床生物学特性和预后的关系，为白血病患者的个体化治疗提供依据，从而提高白血病的疗效，我们进行了以下研究。
HL-60／VCR多药耐药细胞株是研究获得性耐药的一种工具，以往认为其耐药机制是P-gp过度表达。我们采用流式细胞术分别检测了药物敏感细胞株HL-60和多药耐药细胞株HL-60／VCR细胞的耐药蛋白P-gp、MRP、LRP、BCRP、谷胱甘肽-S-转移酶-π(GST-π)以及凋亡调节蛋白bcl-2、bax、bad、bcl-X的表达，结果发现在HL-60／VCR细胞中，耐药蛋白P-gp、MRP、BCRP、GST-π分别是HL-60细胞株的18.62、1.19、1.5、1.32倍，而LRP无变化。抑凋亡蛋白bcl-2、bcl-X分别是其在HL-60细胞株的2.48、1.25倍，调节蛋白bad是HL-60细胞株的1.08倍，而促凋亡蛋白bax反而降低，是HL-60的0.88倍，说明在HL-60／VCR的多药耐药机制中，不但耐药蛋白P-gp、MRP、BCRP、GST-π的表达增强，而且凋亡调节蛋白bcl-2、bax、bcl-X、bad也参与HL60／VCR耐药机制的形成，提示细胞获得性耐药有多种耐药机制参与。
国内已有采用免疫组化或RT-PCR方法对自血病细胞的bcl-2、bax
——
研究，但这些方法难以定量。本研究应用流式细胞术对64例初治AL
患者细胞比上、hcfK、bax、bad的表达进行研究，结果显示：hcf上
在急性髓细胞白血病（AML）的表达较急性淋巴细胞白血病（ALL）
低p1．05）；在AML的各亚型中，bCI上由高到低依次为MI、M4。
M工＊6、m和＊3，后二者分别同前者相比有显著差异01刀5人
bax在＊4中表达较高，与其他类型相比有明显差异01刀5人hcf．
X由高到低依次为 MI。A42、MS、M3、M4，前三者分别明显高于
后二者（P＜0＊5）。bCI－2、bCI－X、b盼及 bsd 的表达在临 床耐药组和
缓解组无显著意义，而冰士儿ax的比值在耐药组明显高于缓解组，
二者相比有统计学意义01．05人根据k上巾ax的比值的大小将AML
分成高危组和低危组：高危组的缓解率为48％，低危组的缓解率为
乃＊％，两组有显著性差异p叱刀5人 提示伙上儿ax的比值影响患
者对化疗的敏感性，是一个独立的预后因素。hcf上／bax比值与k）
均高的患者，缓解率为33．3％，较单独冰上／bax比值高患者的56二％
低，表明比x可使细胞凋亡受抑增加而使白血病患者细胞的耐药性
增强。
我们进一步探讨了比上／bax高比值（）3．73 ）与FAB分型、免
疫表型及细胞遗传学特征在初治成人AML的关系，结果显示：以
M4最高旧0o人 其次为 MS旧1．5oL MI门仰）和 MZ门8．80人
而M3亚型最低，仅占14．3O，M6病例少，有待观察。hcf2巾ax高
比值与CD34表型高度相关（P＜0．05），与CDZ、CD7、CD13、CD14。
CD19和CD33等表面标志无关0功．05人hcf＜／bax高比值在预后
差染色体核型组占62．5％，高于中等预后组的5 7＊％和预后好核型组
的26．3％。提示这些预后因素可通过k上家族调节蛋白来影响化疗
的敏感性。
国内外有关白血病耐药在临床方面的报道，仅少数同时采用多
种耐药因素研究，且多采用免疫组化或RTPCR方法。根据流式细
胞仪具有快速、敏感、精确和定量的特点，我们采用流式细胞术对64
例初发AL患者进行了 P－gp、LRP、GST－。、MRP和 hcf2／bax多
且互
一
种耐药因素的系列临床研究。结果发现：在AML患者中（M3除外人
临床耐药组中P－gP、LRP、bclZ／bax均较临床缓解组中高；而GST－
卜MRP在临床耐药组虽比缓解组高，但无统计学意义0叩刀5人
患者P－gp、LRP和 bclZ／bax的阳性率为 29．2％、37．5％和 52．l％，卜
性组患者的缓解率为 28石％、44．4％和 48％，分别较阴性组患者的
＊．＊％、刀．3％禾78二％低（P＜O刀5）。MRP和＊盯－。的阳性率为31二％
和 50o，其缓解率分别为 46，60和 500，与阴性组相比无显著差异
o＞0刀5人 在临床耐药组中，绝大多数患者多种耐药因素共存，18
例患者中涉及 4种、3种和 2种耐药因素的分别有 2例、门例和 5
例。卞反在缓解组中，30例中仅5例患者有3禾和 12例患者有2吞
耐药因素共存。表明在临床原发耐药患者中，大多有多种耐药因素
参与其细胞耐药的形成，它们共同作用的结果，最终导致细胞耐药。
这也解释了为什么临床上卜gp阳性的难治患者在化疗同时应用卜gp
逆转剂疗效差、以及为什么部分卜gp阴性患者预后也差的原因。我
们发现7例M3 i者其耐药因素明显较少，这可能是M3患者治疗效
果好的重要原因。
BCny是 1998年发现的一种耐药蛋白，在 AL中的报道甚少。
The chemotherapy is the major and basic method to treat leukemia, and also the basis of the stem cell transplantation. During recent years, although many new chemotherapeutic drugs and protocols have been developed to treat leukemia, most patients ultimately died of treatment failure. There are many possibilities of the failure and drug resistance may be the most important factor. Many mechanisms can result in cellular multidrug resistance (MDR) including P-glycoprotein (P-gp), multidrug resistance associated protein (MRP),lung resistance protein(LRP) and breast cancer resistance protein(BCRP) which served as drug "pump" or "entrapment", altered apoptosis mechanisms, as well as enzymatic drug detoxification and so on. The following experiments were performed to investigate comprehensively the expression of drug resistance proteins and apoptosis modulating protein, and their relationship with the biological characters and prognosis in patients with acute- leukemia. That may provide the guide to tailor chemoth
erapy for individual patients and increase chemotherapeutic effect.
HL-60/VCR cells were used as a good model to study acquired chemoresistance. The previous data showed that the mechanism of MDR was the overexpression of P-gp.In this study, flow cytometry was used to analyze the expression of MDR proteins including P-gp, MRP, LRP, BCRP and GST- n , apoptosis modulating proteins including bcl-2, bcl-X, bax and bad in HL-60/VCR and HL-60. The results showed that the expression of P-gp, MRP, BCRP, GST- n were 18.62, 1.19, 1.5, 1.32 -fold
in HL-60/VCR as much as that in HL-60,respectiveIy, but LRP was not different between the two cell lines. The apoptosis modulating proteins of bcl-2, bcl-X, bad were 2.48, 1.25, 1.08 -fold in HL-60/VCR as much as in HL-60, respectively, while the pro-apoptosis protein bax decreased to 0.88-fold as mush as in HL-60. This study demonstrated that various MDR mechanisms were involved in HL-60/VCR,which including P-gp, MRP, BCRP and GST- n , the apoptosis modulating proteins bcl-2, bcl-X, bax and bad, and suggested that acquired chemoresistance were caused by several drug resistance mechanisms.
The immunohistochemistry and RT-PCR have been used to measure the expression of bcl-2,bax in acute leukemia. In this study,flow cytometry was used to investigate the expression of bcl-2, bcl-X, bax, bad in 64 patients with de novo acute leukemia. We found that the expression of bcl-2 in acute myeloid leukemia(AML) was lower than that in acute lymphoid leukemia(ALL) (P<0.05). In AML subtypes, the expression of bcl-2 in Ml, M4 and M5 was significantly higher than that in M2, M3 (P<0.05), respectively. The expression of bax was higher in M4 than that in other AML subtypes (PO.05). The expression of bcl-X was higher in Ml, M2 M5 than that in M3, M4 (PO.05), respectively. The expression of bcl-2, bcl-X, bax and bad was not significantly different between chemoresistant and complete remission(CR) patients, respectively, but the ratio of bcl-2/bax was significantly higher in chemoresistant patients than that in patients achieving CR (PO.05). The CR rate was 48% in the high-risk group, which was significantly lower than 78.2% in the low-risk group (PO.05). It suggests that the ratio of bcl-2/bax may influence the therapeutic sensibility, and it is an independent prognostic factor in de novo AML. The CR rate was 33.3% in patients with high ratio of bcl-2/bax and high expression of bcl-X, which was lower than that in the patients with high ratio of bcl-2/bax and low expression of bcl-X (56.2%).
Our study suggests that bcl-X might reinforce the ability of anti-apoptosis and increase the drug resistance.
We further explored the correlation of the high ratio of bcl-2/bax with clinical and biological characteristics in de novo AML. We found that the high ratio of bcl-2/bax was more frequent in M4(80%),and then in M5(61.5%),M1(50%) and M2(38.8%),the lowest in M3(14.3%). The high ratio of bcl-2/bax was significantly associated with CD34 expression (P<0.05), and had no correlation with CD2, CD
HL-60／VCR多药耐药细胞株是研究获得性耐药的一种工具，以往认为其耐药机制是P-gp过度表达。我们采用流式细胞术分别检测了药物敏感细胞株HL-60和多药耐药细胞株HL-60／VCR细胞的耐药蛋白P-gp、MRP、LRP、BCRP、谷胱甘肽-S-转移酶-π(GST-π)以及凋亡调节蛋白bcl-2、bax、bad、bcl-X的表达，结果发现在HL-60／VCR细胞中，耐药蛋白P-gp、MRP、BCRP、GST-π分别是HL-60细胞株的18.62、1.19、1.5、1.32倍，而LRP无变化。抑凋亡蛋白bcl-2、bcl-X分别是其在HL-60细胞株的2.48、1.25倍，调节蛋白bad是HL-60细胞株的1.08倍，而促凋亡蛋白bax反而降低，是HL-60的0.88倍，说明在HL-60／VCR的多药耐药机制中，不但耐药蛋白P-gp、MRP、BCRP、GST-π的表达增强，而且凋亡调节蛋白bcl-2、bax、bcl-X、bad也参与HL60／VCR耐药机制的形成，提示细胞获得性耐药有多种耐药机制参与。
国内已有采用免疫组化或RT-PCR方法对自血病细胞的bcl-2、bax
——
研究，但这些方法难以定量。本研究应用流式细胞术对64例初治AL
患者细胞比上、hcfK、bax、bad的表达进行研究，结果显示：hcf上
在急性髓细胞白血病（AML）的表达较急性淋巴细胞白血病（ALL）
低p1．05）；在AML的各亚型中，bCI上由高到低依次为MI、M4。
M工＊6、m和＊3，后二者分别同前者相比有显著差异01刀5人
bax在＊4中表达较高，与其他类型相比有明显差异01刀5人hcf．
X由高到低依次为 MI。A42、MS、M3、M4，前三者分别明显高于
后二者（P＜0＊5）。bCI－2、bCI－X、b盼及 bsd 的表达在临 床耐药组和
缓解组无显著意义，而冰士儿ax的比值在耐药组明显高于缓解组，
二者相比有统计学意义01．05人根据k上巾ax的比值的大小将AML
分成高危组和低危组：高危组的缓解率为48％，低危组的缓解率为
乃＊％，两组有显著性差异p叱刀5人 提示伙上儿ax的比值影响患
者对化疗的敏感性，是一个独立的预后因素。hcf上／bax比值与k）
均高的患者，缓解率为33．3％，较单独冰上／bax比值高患者的56二％
低，表明比x可使细胞凋亡受抑增加而使白血病患者细胞的耐药性
增强。
我们进一步探讨了比上／bax高比值（）3．73 ）与FAB分型、免
疫表型及细胞遗传学特征在初治成人AML的关系，结果显示：以
M4最高旧0o人 其次为 MS旧1．5oL MI门仰）和 MZ门8．80人
而M3亚型最低，仅占14．3O，M6病例少，有待观察。hcf2巾ax高
比值与CD34表型高度相关（P＜0．05），与CDZ、CD7、CD13、CD14。
CD19和CD33等表面标志无关0功．05人hcf＜／bax高比值在预后
差染色体核型组占62．5％，高于中等预后组的5 7＊％和预后好核型组
的26．3％。提示这些预后因素可通过k上家族调节蛋白来影响化疗
的敏感性。
国内外有关白血病耐药在临床方面的报道，仅少数同时采用多
种耐药因素研究，且多采用免疫组化或RTPCR方法。根据流式细
胞仪具有快速、敏感、精确和定量的特点，我们采用流式细胞术对64
例初发AL患者进行了 P－gp、LRP、GST－。、MRP和 hcf2／bax多
且互
一
种耐药因素的系列临床研究。结果发现：在AML患者中（M3除外人
临床耐药组中P－gP、LRP、bclZ／bax均较临床缓解组中高；而GST－
卜MRP在临床耐药组虽比缓解组高，但无统计学意义0叩刀5人
患者P－gp、LRP和 bclZ／bax的阳性率为 29．2％、37．5％和 52．l％，卜
性组患者的缓解率为 28石％、44．4％和 48％，分别较阴性组患者的
＊．＊％、刀．3％禾78二％低（P＜O刀5）。MRP和＊盯－。的阳性率为31二％
和 50o，其缓解率分别为 46，60和 500，与阴性组相比无显著差异
o＞0刀5人 在临床耐药组中，绝大多数患者多种耐药因素共存，18
例患者中涉及 4种、3种和 2种耐药因素的分别有 2例、门例和 5
例。卞反在缓解组中，30例中仅5例患者有3禾和 12例患者有2吞
耐药因素共存。表明在临床原发耐药患者中，大多有多种耐药因素
参与其细胞耐药的形成，它们共同作用的结果，最终导致细胞耐药。
这也解释了为什么临床上卜gp阳性的难治患者在化疗同时应用卜gp
逆转剂疗效差、以及为什么部分卜gp阴性患者预后也差的原因。我
们发现7例M3 i者其耐药因素明显较少，这可能是M3患者治疗效
果好的重要原因。
BCny是 1998年发现的一种耐药蛋白，在 AL中的报道甚少。
The chemotherapy is the major and basic method to treat leukemia, and also the basis of the stem cell transplantation. During recent years, although many new chemotherapeutic drugs and protocols have been developed to treat leukemia, most patients ultimately died of treatment failure. There are many possibilities of the failure and drug resistance may be the most important factor. Many mechanisms can result in cellular multidrug resistance (MDR) including P-glycoprotein (P-gp), multidrug resistance associated protein (MRP),lung resistance protein(LRP) and breast cancer resistance protein(BCRP) which served as drug "pump" or "entrapment", altered apoptosis mechanisms, as well as enzymatic drug detoxification and so on. The following experiments were performed to investigate comprehensively the expression of drug resistance proteins and apoptosis modulating protein, and their relationship with the biological characters and prognosis in patients with acute- leukemia. That may provide the guide to tailor chemoth
erapy for individual patients and increase chemotherapeutic effect.
HL-60/VCR cells were used as a good model to study acquired chemoresistance. The previous data showed that the mechanism of MDR was the overexpression of P-gp.In this study, flow cytometry was used to analyze the expression of MDR proteins including P-gp, MRP, LRP, BCRP and GST- n , apoptosis modulating proteins including bcl-2, bcl-X, bax and bad in HL-60/VCR and HL-60. The results showed that the expression of P-gp, MRP, BCRP, GST- n were 18.62, 1.19, 1.5, 1.32 -fold
in HL-60/VCR as much as that in HL-60,respectiveIy, but LRP was not different between the two cell lines. The apoptosis modulating proteins of bcl-2, bcl-X, bad were 2.48, 1.25, 1.08 -fold in HL-60/VCR as much as in HL-60, respectively, while the pro-apoptosis protein bax decreased to 0.88-fold as mush as in HL-60. This study demonstrated that various MDR mechanisms were involved in HL-60/VCR,which including P-gp, MRP, BCRP and GST- n , the apoptosis modulating proteins bcl-2, bcl-X, bax and bad, and suggested that acquired chemoresistance were caused by several drug resistance mechanisms.
The immunohistochemistry and RT-PCR have been used to measure the expression of bcl-2,bax in acute leukemia. In this study,flow cytometry was used to investigate the expression of bcl-2, bcl-X, bax, bad in 64 patients with de novo acute leukemia. We found that the expression of bcl-2 in acute myeloid leukemia(AML) was lower than that in acute lymphoid leukemia(ALL) (P<0.05). In AML subtypes, the expression of bcl-2 in Ml, M4 and M5 was significantly higher than that in M2, M3 (P<0.05), respectively. The expression of bax was higher in M4 than that in other AML subtypes (PO.05). The expression of bcl-X was higher in Ml, M2 M5 than that in M3, M4 (PO.05), respectively. The expression of bcl-2, bcl-X, bax and bad was not significantly different between chemoresistant and complete remission(CR) patients, respectively, but the ratio of bcl-2/bax was significantly higher in chemoresistant patients than that in patients achieving CR (PO.05). The CR rate was 48% in the high-risk group, which was significantly lower than 78.2% in the low-risk group (PO.05). It suggests that the ratio of bcl-2/bax may influence the therapeutic sensibility, and it is an independent prognostic factor in de novo AML. The CR rate was 33.3% in patients with high ratio of bcl-2/bax and high expression of bcl-X, which was lower than that in the patients with high ratio of bcl-2/bax and low expression of bcl-X (56.2%).
Our study suggests that bcl-X might reinforce the ability of anti-apoptosis and increase the drug resistance.
We further explored the correlation of the high ratio of bcl-2/bax with clinical and biological characteristics in de novo AML. We found that the high ratio of bcl-2/bax was more frequent in M4(80%),and then in M5(61.5%),M1(50%) and M2(38.8%),the lowest in M3(14.3%). The high ratio of bcl-2/bax was significantly associated with CD34 expression (P<0.05), and had no correlation with CD2, CD
引文
1 Arceci RJ.Can multidrug resistance mechanisms be modified? Br J Haematol, 2000, 110. 285-291
2 竺香才，郭鹃，王荷碧，等．人类白血病耐药细胞系K562/VCR mdrl及GST表达的研究．中华血液学杂志，1995，16：193-194
3 夏瑞祥，林果为，励雁蜂，等．耐药细胞系GSH含量，GST活力及同工酶，MRP表达的研究．中华血液学杂志，1998，19：247-248
4 Huang Y,Ibrado AM,Reed JC,et al.Co-expression of several molecular mechanisms of multidrug resistance and their significance for paclitaxel cytotoxity in human AML,HL-60 cells.Leukemia,1997,11:253-257
5 Solary E,Droin N,Corcos L,et al. Positive and negative regulation of apoptotic pathways by cytotoxic agents in hematological malignancies. Leukemia,2000,14:1833-1849
6 Campos L,Rouault JP,Sabido O,et al.High expression of bcl-2 protein in acute myeloid-leukemia cells is associated with poor response to chemotherapy.Blood,1993,81:3091-3099
7 Maung ZT.The relationship between bcl-2 expression and response to chemotherapy in acute leukemia. Br J Haematol,1994,88:105-110
8 Kornblau SM,Vu HT,Ruvolo P,et al. Bax and PKCalpha modulate the prognostic impact of bcl-2 expression in acute myelogenous leukemia.Clin Cancer Res,2000,6:1401-1409
9 Pallis M,Zhu YM,Russell NH. Bcl-XL is heterogeneously expressed by acute P-glycoprotein expression. Leukemia,1997,11:945-949
10 Filipits M,Stranzl T,Pohl G,et al.Drug resistance factors in acute myeloid leukemia: a comparative analysis.Leukemia,2000,14:68-76
11 Lohri A,van Hille B,Bacchi M,et al.Five putative drug resistance parameters (MDR 1/P-glycoprotein,MDR-associated protein,
glutathione-S-transferase,bcl-2 and topoisomerase Ⅱa)in 57 newly diagnosed acute myeloid leukaemias.Eur I Haematol 1997,59:206-215
12 上海市白血病协作组．急性白血病患者P-gp,mdrl,MRP和TopoⅡ表达及其预后关系的研究．中华血液学杂志．2001，22：90-93
13 Douglas D,Ross D,Judith K,et al.Expression of breast cancer resistance protein in blast cells from patients With acute leukemia.Blood,2000;96:365-369
2 竺香才，郭鹃，王荷碧，等．人类白血病耐药细胞系K562/VCR mdrl及GST表达的研究．中华血液学杂志，1995，16：193-194
3 夏瑞祥，林果为，励雁蜂，等．耐药细胞系GSH含量，GST活力及同工酶，MRP表达的研究．中华血液学杂志，1998，19：247-248
4 Huang Y,Ibrado AM,Reed JC,et al.Co-expression of several molecular mechanisms of multidrug resistance and their significance for paclitaxel cytotoxity in human AML,HL-60 cells.Leukemia,1997,11:253-257
5 Solary E,Droin N,Corcos L,et al. Positive and negative regulation of apoptotic pathways by cytotoxic agents in hematological malignancies. Leukemia,2000,14:1833-1849
6 Campos L,Rouault JP,Sabido O,et al.High expression of bcl-2 protein in acute myeloid-leukemia cells is associated with poor response to chemotherapy.Blood,1993,81:3091-3099
7 Maung ZT.The relationship between bcl-2 expression and response to chemotherapy in acute leukemia. Br J Haematol,1994,88:105-110
8 Kornblau SM,Vu HT,Ruvolo P,et al. Bax and PKCalpha modulate the prognostic impact of bcl-2 expression in acute myelogenous leukemia.Clin Cancer Res,2000,6:1401-1409
9 Pallis M,Zhu YM,Russell NH. Bcl-XL is heterogeneously expressed by acute P-glycoprotein expression. Leukemia,1997,11:945-949
10 Filipits M,Stranzl T,Pohl G,et al.Drug resistance factors in acute myeloid leukemia: a comparative analysis.Leukemia,2000,14:68-76
11 Lohri A,van Hille B,Bacchi M,et al.Five putative drug resistance parameters (MDR 1/P-glycoprotein,MDR-associated protein,
glutathione-S-transferase,bcl-2 and topoisomerase Ⅱa)in 57 newly diagnosed acute myeloid leukaemias.Eur I Haematol 1997,59:206-215
12 上海市白血病协作组．急性白血病患者P-gp,mdrl,MRP和TopoⅡ表达及其预后关系的研究．中华血液学杂志．2001，22：90-93
13 Douglas D,Ross D,Judith K,et al.Expression of breast cancer resistance protein in blast cells from patients With acute leukemia.Blood,2000;96:365-369