急性白血病耐药相关基因表达及其与临床关系的研究
|
摘要
目的 为较系统全面地探讨急性白血病耐药相关基因与临床化疗疗效、预后的关系，本文应用半定量和(或)定量逆转录—聚合酶链反应(Reverse transcription polymerase chain reaction，RT-PCR)检测急性白血病患者多药耐药基因(Multidrug resistance gene-1，mdr-1)、多药耐药相关蛋白基因(Multidrug resistance-associated protein gene，mrp)、肺耐药相关蛋白基因(Lung resistance-related protein gene，1rp)、乳腺癌耐药相关蛋白基因(Breast cancer resistance protein gene，bcrp)、谷胱苷肽S转移酶基因(Glutathione-S-transferases，GSTs)、DNA拓扑异构酶基因(Topoisomerase，Topo)及bcl-2基因表达水平的高低，为判断急性白血病多药耐药(Multidrugresistance，MDR)的发生和预后、克服白血病MDR提供实验依据。
方法 将140例急性白血病(Acute Leukemia，AL)患者按病程分为初治组、完全缓解组和复发组；按治疗效果分为敏感组和耐药组。用半定量RT-PCR法检测急性白血病患者外周血或骨髓中bcrp、mdr-1、mrp、1rp、GSTα、GSTπ、TopoⅠ、TopoⅡα、TopoⅡβ、bcl-2等基因的mRNA表达水平的高低。通过琼脂凝胶电泳法，计算
有关基因的相对表达量。还建立实时荧光定量 RTICR（Real time
luorescent quantitative RT－PCR，FQ RT－PCR）检坝 bcrp、mdr－l、p。
lrp的精确拷贝数，探讨化疗疗效及预后与耐药相关基因表达之间的
关系。
结果（一）通过半定量 RTPCR检测 AL患者上述 10个基因
的表达水平，得到如下结果：
1．AL组 bcrp、lrp、GST厂、TOpo 11a、Tope 11日十对表达水
平明显高于对照组，GST Q表达水平明显低于对照组（P＜0刀引。
2．AL复发组bcrp、md，刁表达水平Hg显高于初治组，差别具有
显著性（ ＜0刀1）。
3．AL耐药组mdr叫表达水平明显高于敏感组 巾＜0刀引。
4．耐药组bcrp、mdr刁、mrp表达阳性率明显高于敏感组（P＜0刀引。
5．bcrp、mdr叫、mrp、lrp基因同时阳性的思者CR率明显低于
1～3个基因阳性的患者 中刃刀1人
6．经等级柏关分析显示 mdr！ GST。与耐药性呈显著正相关，
GST。和 TopollQ与耐药性呈显著负相关（P＜0＊引。
7．经多因素逻辑回归分析示ndr刁与预后不良呈显著正相关。
8．经直线相关分析，bcrp与mdr八 mdrl与lrp、mdrJ与GST
a、mrp与 lrp呈显著正相关，P＜0刀5。m甲与 GST。呈显著负相关，
P＜0刀1。
（二）为了更准确测定 bcrp、mdr刁、mrp、冲基因的拷贝数，
我们建立了 FQ RTPCR方法，制备了标准曲线，结果显示：
3
1．AL组krp基因拷贝数明显高于对照组0叼．05人
2．从复发组mdrl拷贝数明显高于初治组0＜0刀引。
3．AL耐药组 mdrl拷贝数明显高于敏感组o＜0刀5入
4，经直线相关分析显示mdrq与帅、mdrl与冲、p与一
呈显著正相关。
5．经等级相关分祈示mdr刁和冲的表达与耐药性密切相关。
结论 通过本研究结果得出如下结论：l．急性白血病多药耐药的
发生涉及的机制较多，与bcrp、mdr刁 mrp及lrp基因中一项或几项
异常表达有关，bcrp、mdr刁、m叩及lrp均可作为判断临床多药耐药
的指标。2．GSTS和 TopO在酶介导耐药机制中的具体作用需结合其它
因素综合分祈，不能将其孤立地作为判断耐药的肯定指标。3．多基因
联合运用预测临床MDR效果优于单一基因的预测，白血病多药耐药
的发生是多种基因共同作用的结果。但仅从上还有关基因的检测仍不
能完全解释临床发主耐药的情况，可能存在其他未知的新的耐药机
制。
Objective To better investigate and understand the relationship between the drug-resistance related genes and chemotherapy response, prognosis in acute leukemia (AL), semi-quantitative and real time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of the multidrug resistance gene-1 (mdr-1), multidrug resistance-associated protein gene (mrp), lung resistance-related protein gene (Irp), breast cancer resistance protein gene (bcrp), Glutathione S-transferases (GSTs), DNA Topoisomerase (Topo) and bcl-2 genes from patients with AL, from which experimental data for judging multidrug resistance (MDR) and clinical prognosis would be provided.
Method 140 AL patients were divided into three groups: untreated (newly diagnosed) > complete remission (CR) and relapsed patients. In addition, sensitive and resistance groups were also designated
according to chemotherapy response. The expression level of the mdr-1 ,mrp, lip, bcrp ,GST a , GST ft, Topo I ,Topo II a JOpo II ?, bcl-2 genes were detected by semi-qantitative RT-PCR in peripheral blood or bone marrow sample from patients. After agarose gel electrophoresis, the relative expression level was calculated as related genes/ 0 2-MG or |3 -actin ratio. We also established more accurate real time fluorescent quantitative RT-PCR with TaqMan (FQ RT-PCR) to detect accurate mRNA copies of bcrp, mdr-1, mrp and lip.
Results The results from semi-quantitative RT-PCR showed:
I .The relative expression level of bcrp, lip, GST ft , Topo II a and Topo
II 3 in AL group were significantly higher than that in normal subjects. GST a , however, was exactly reverse (p<0.05). 2.The expression of bcrp and mdr-1 was significantly higher in relapsed group comparing with untreated group. 3. Resistance group had higher mdr-1 levels than sensitive group. 4.The positive percentage of bcrp, mdr-1 and mrp expression in resistance group were higher than that in sensitive group (p<0.05). 5. Patients who overexpressed bcrp, mdr-1, imp and lip at the same time had a poorer response and lower CR rate. 6.Rank correlation analysis showed that overexpression of mdr-1 and GST a were highly correlated with clinical drug resistance. It was also found that there were significantly negative correlation between GST ft , Topo II a and clinical resistance. 7. Multivariable logistic regression analysis showed that mdr-1
had a positive correlation with poor prognosis (p<0.05). 8. With linear correlation analysis, positive correlation were observed between bcrp and mdr-1, mdr-1 and Irp, mdr-1 and GST a , mrp and Irp; negative correlation were observed between mrp and GST n (p<0.05).
In order to measure the copies of bcrp, mdr-1, mrp and lip mRNA more accurately, we established FQ RT-PCR. The result showed: 1. Copies of bcrp mRNA in AL group were significantly higher than that in normal one (p<0.05). 2. Copies of mdr-1 mRNA in relapsed groups were significantly higher than that in normal one (p<0.05). 3. Resistance group had significantly higher copies of mdr-1 mRNA than that in sensitive group. 4. Positive correlations were observed between mdr-1 and mrp, mdr-1 and Irp, mrp and Irp. 5. Expression level of mdr-1 and Irp were significantly correlated with clinical resistance through rank correlation analysis.
Conclusion Through the study mentioned above, we can draw following conclusions: l.Overexpressions of one or more genes of bcrp, mdr-1, mrp and Irp are associated with MDR in AL. Bcrp, mdr-1, mrp and Irp can be the indicators for judging MDR. 2. To clarify the roles of GSTs and Topo in MDR, other factors or variables should be considered. Neither GSTs nor Topo alone is a good indicator for MDR. 3. In evaluation of MDR the combined detection of multi-genes are better than that of single gene. Occurance of MDR is the consequence of multiple
mechanisms. Drug resistance could not be explain by the genes mentioned'above, new MDR mechanism remains to be explored.
方法 将140例急性白血病(Acute Leukemia，AL)患者按病程分为初治组、完全缓解组和复发组；按治疗效果分为敏感组和耐药组。用半定量RT-PCR法检测急性白血病患者外周血或骨髓中bcrp、mdr-1、mrp、1rp、GSTα、GSTπ、TopoⅠ、TopoⅡα、TopoⅡβ、bcl-2等基因的mRNA表达水平的高低。通过琼脂凝胶电泳法，计算
有关基因的相对表达量。还建立实时荧光定量 RTICR（Real time
luorescent quantitative RT－PCR，FQ RT－PCR）检坝 bcrp、mdr－l、p。
lrp的精确拷贝数，探讨化疗疗效及预后与耐药相关基因表达之间的
关系。
结果（一）通过半定量 RTPCR检测 AL患者上述 10个基因
的表达水平，得到如下结果：
1．AL组 bcrp、lrp、GST厂、TOpo 11a、Tope 11日十对表达水
平明显高于对照组，GST Q表达水平明显低于对照组（P＜0刀引。
2．AL复发组bcrp、md，刁表达水平Hg显高于初治组，差别具有
显著性（ ＜0刀1）。
3．AL耐药组mdr叫表达水平明显高于敏感组 巾＜0刀引。
4．耐药组bcrp、mdr刁、mrp表达阳性率明显高于敏感组（P＜0刀引。
5．bcrp、mdr叫、mrp、lrp基因同时阳性的思者CR率明显低于
1～3个基因阳性的患者 中刃刀1人
6．经等级柏关分析显示 mdr！ GST。与耐药性呈显著正相关，
GST。和 TopollQ与耐药性呈显著负相关（P＜0＊引。
7．经多因素逻辑回归分析示ndr刁与预后不良呈显著正相关。
8．经直线相关分析，bcrp与mdr八 mdrl与lrp、mdrJ与GST
a、mrp与 lrp呈显著正相关，P＜0刀5。m甲与 GST。呈显著负相关，
P＜0刀1。
（二）为了更准确测定 bcrp、mdr刁、mrp、冲基因的拷贝数，
我们建立了 FQ RTPCR方法，制备了标准曲线，结果显示：
3
1．AL组krp基因拷贝数明显高于对照组0叼．05人
2．从复发组mdrl拷贝数明显高于初治组0＜0刀引。
3．AL耐药组 mdrl拷贝数明显高于敏感组o＜0刀5入
4，经直线相关分析显示mdrq与帅、mdrl与冲、p与一
呈显著正相关。
5．经等级相关分祈示mdr刁和冲的表达与耐药性密切相关。
结论 通过本研究结果得出如下结论：l．急性白血病多药耐药的
发生涉及的机制较多，与bcrp、mdr刁 mrp及lrp基因中一项或几项
异常表达有关，bcrp、mdr刁、m叩及lrp均可作为判断临床多药耐药
的指标。2．GSTS和 TopO在酶介导耐药机制中的具体作用需结合其它
因素综合分祈，不能将其孤立地作为判断耐药的肯定指标。3．多基因
联合运用预测临床MDR效果优于单一基因的预测，白血病多药耐药
的发生是多种基因共同作用的结果。但仅从上还有关基因的检测仍不
能完全解释临床发主耐药的情况，可能存在其他未知的新的耐药机
制。
Objective To better investigate and understand the relationship between the drug-resistance related genes and chemotherapy response, prognosis in acute leukemia (AL), semi-quantitative and real time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of the multidrug resistance gene-1 (mdr-1), multidrug resistance-associated protein gene (mrp), lung resistance-related protein gene (Irp), breast cancer resistance protein gene (bcrp), Glutathione S-transferases (GSTs), DNA Topoisomerase (Topo) and bcl-2 genes from patients with AL, from which experimental data for judging multidrug resistance (MDR) and clinical prognosis would be provided.
Method 140 AL patients were divided into three groups: untreated (newly diagnosed) > complete remission (CR) and relapsed patients. In addition, sensitive and resistance groups were also designated
according to chemotherapy response. The expression level of the mdr-1 ,mrp, lip, bcrp ,GST a , GST ft, Topo I ,Topo II a JOpo II ?, bcl-2 genes were detected by semi-qantitative RT-PCR in peripheral blood or bone marrow sample from patients. After agarose gel electrophoresis, the relative expression level was calculated as related genes/ 0 2-MG or |3 -actin ratio. We also established more accurate real time fluorescent quantitative RT-PCR with TaqMan (FQ RT-PCR) to detect accurate mRNA copies of bcrp, mdr-1, mrp and lip.
Results The results from semi-quantitative RT-PCR showed:
I .The relative expression level of bcrp, lip, GST ft , Topo II a and Topo
II 3 in AL group were significantly higher than that in normal subjects. GST a , however, was exactly reverse (p<0.05). 2.The expression of bcrp and mdr-1 was significantly higher in relapsed group comparing with untreated group. 3. Resistance group had higher mdr-1 levels than sensitive group. 4.The positive percentage of bcrp, mdr-1 and mrp expression in resistance group were higher than that in sensitive group (p<0.05). 5. Patients who overexpressed bcrp, mdr-1, imp and lip at the same time had a poorer response and lower CR rate. 6.Rank correlation analysis showed that overexpression of mdr-1 and GST a were highly correlated with clinical drug resistance. It was also found that there were significantly negative correlation between GST ft , Topo II a and clinical resistance. 7. Multivariable logistic regression analysis showed that mdr-1
had a positive correlation with poor prognosis (p<0.05). 8. With linear correlation analysis, positive correlation were observed between bcrp and mdr-1, mdr-1 and Irp, mdr-1 and GST a , mrp and Irp; negative correlation were observed between mrp and GST n (p<0.05).
In order to measure the copies of bcrp, mdr-1, mrp and lip mRNA more accurately, we established FQ RT-PCR. The result showed: 1. Copies of bcrp mRNA in AL group were significantly higher than that in normal one (p<0.05). 2. Copies of mdr-1 mRNA in relapsed groups were significantly higher than that in normal one (p<0.05). 3. Resistance group had significantly higher copies of mdr-1 mRNA than that in sensitive group. 4. Positive correlations were observed between mdr-1 and mrp, mdr-1 and Irp, mrp and Irp. 5. Expression level of mdr-1 and Irp were significantly correlated with clinical resistance through rank correlation analysis.
Conclusion Through the study mentioned above, we can draw following conclusions: l.Overexpressions of one or more genes of bcrp, mdr-1, mrp and Irp are associated with MDR in AL. Bcrp, mdr-1, mrp and Irp can be the indicators for judging MDR. 2. To clarify the roles of GSTs and Topo in MDR, other factors or variables should be considered. Neither GSTs nor Topo alone is a good indicator for MDR. 3. In evaluation of MDR the combined detection of multi-genes are better than that of single gene. Occurance of MDR is the consequence of multiple
mechanisms. Drug resistance could not be explain by the genes mentioned'above, new MDR mechanism remains to be explored.
引文
1． Lemontt IF, Azzaria M, Gros P. Increased mdr gene expression and decreased drug accumulation in multidrug-resistance human melanoma cell. Cancer Res. 1988;48:6348-6353．
2． Fojo AT. Whang-Peng J, Gottesmann MM, Pastan I. Amplification of DNA sequences in human multidrug-resistance KB carcinoma cells. Proc Natl Acad Sci. USA. 1985;82:7661-7665．
3． Cole SPC. Bhardwaj G, Gerlach JH, et al. Overexpression of a transporter gene in a multidrug-resistance human lung cerner cell line. Science. 1992,258:1050-1664．
4． Scheper RJ, Broxterman HJ, Scheffer GL, et al. Overexpression of a M(r) 110,000 vesicular protein in non-P-glycoprotein-mediated multidrug resistance. Cancer Res. 1993;53:1475-1479．
5． Doyle L A, Yang Werdong, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Prol Natl Acad Sci. USA. 1998;95:15665-15670．
6． Lorico A, Rappa G, Srimatkandada S, et al. Increased rate of adenosine triphosphate-dependent etoposide (VP-16) efflux in a murine leukemia cell line overexpressing the multidrug resistomce-associated protein (MRP) gene. Cancer Res. 1995;55:4352-4360．
7． Patskovsky YY, Patskovska LN, Listowsky I, et al. Functions of His 107 in the catalytic mechanism of human glutathione s-transferase hGSTMla-la. Biochemistry. 1999;38:1193-1202．
8． Agular-Santelises M, Rottenberg ME, et al. Bcl-2, Bax and P53 expression in B-CLL in relation to in vitro survival and clinical progression. Int J Cancer. 1996;69:114-119．
9． 上海市白血病协作组，急性白血病患者P-gp，mdr-1，MRP和Topo II表达及其与预后关系的研究。中华血液学杂志，2001；22(2) ：90-93．
10． Arceci RJ. Clinical significance of P-glycoprotein in multidrug resistance malignancies. Blood. 1993;81:2215-2222．
11． Nooter K, Herweijer H. Multidrug resistance (mdr) genes in human cancer. Br J Cancer. 1991;63:663-669．
12． Cole SPC, Bhardwaj G, Gerlach JH. et al. Overexpression of a transporter gene in a multidrug-resistance human lung cancer cell line Science. 1992;258:1650-1664．
13． Nooter K, Burger H, Stoter G. Multidrug resistance-associated protein (MRP) in haematological malignancies. Leuk lymphoma. 1995;20:381-387．
14． Zhou DC, Zittoun R, Marie JP. Expression of the multidrug resistance-associated protein (MRP) and multidrug resistance (mdr-1) genes in acute myeloid leukemia. Leukemia. 1995;9:1661-1666．
15． Erasmus Schneider, Kenneth H, et al. Increased expression of the
multidrng resistance-associated protein gene in relapsed acute leukemia. Blood. 1995; 85: 186-193．
16． Herman Burger, Kees Nooter, et al. Expression of the multidrug resistance-associated protein (MRP) in acute and chronic leukemia. Leukemia. 1994;8:990-997．
17． Hart SM, GaneShaguru K, et al. Expression of the multidrug resistance-associated protein (MRP) in acute leukemia. Leukemia. 1994;8:2163-2168．
18． Hart SM, Ganeshaguru K, Scheper RJ, et al. Expression of the human major vault protein LRP in acute myeloid leukemia. Exp Hematol. 1997;25:1227-1232．
19． Rocchi E, Khodjakov A, Volk EL. et al. The product of the ABC half-transporter gene ABCG2(BCRP/MXR/ABCR) is expressed in the plasma membrane. Biochem Biophys Res commum. 2000;27 1:42-46．
20． Ross DD, Karp JE, Chen TT, et al. Expression of breast cancer resistance protein in blast cells from patients with acute leukemia. Blood. 2000,96:365-368．
21． 郑德先，吴克复，褚建新等．现代实验血液学研究方法与技术。 北京医科大学、中国协和医院大学联合出版社．1999． 4，第十版
22． Morrow CS, Cowan KH, Goldsmith ME, et al. Structure of the human genomic glutathione s-transferase-pi gene. Gene. 1989;75:3-11．
23． Tiirikainen MI, Elonen E, Syrjala MT, et al. Flow cytometric analysis
of glutathione S-transferase π in acute leukemia. Leukemia. 1994;8:978-984．
24． Sinha BK, Katki AG, Batist G, et al. Differential formation of hydroxyl radicals by adriamycin in sensitive and resistant MCF-7 human breast tumor cells implication for the mechanism of action. Biochemistry. 1987;26:3776-3781．
25． Litwack G, Kettever B, Arias LM, et al. Ligandin: A hepatic protein which binds steroids, bilirubin, carcinogens and a number of exogenous organic anions. Nature. 1997;234:466-467．
26． Massaad-Massade L, Ribrag V, Marie JP, et al. Glutathione system, topoisomerase Ⅱ level and multidrug resistance phenotype in acute myelogenous leukemia before treatment and at relapse. Anticancer Res. 1997;17:4647-4651．
27． Den Boer ML, Pieters R, et al. Different expression of glutathione S-transferase a , μ and π in childhood acute lymphoblastic and myeloid leukemia. Br J Haematol. 1999,104:321-327．
28． Rosario LA, O' Brien ML, Henderson CJ, et al. Cellular response to a glutathione s-transferase P1-1 activated prodrug. Mol Pharmacol, 2000;58:167-174．
29． Withoff S, Jong De S, Vries De EG, et al. Humanal DNA Topoisomerase Ⅱ: Biochemistry and role in chemotherapy resistance. Anticancer Res. 1996;16:1867-1870．
30． Beck J, Handgretinger R, Klingebiel T, et al. Expression of PKC isozyme and MDR-associated genes in primary and relapsed state. AML. Leukemia. 1996; 10:426-433．
31． Gieseler F, Glasmacher A, Kampfe D, et al. Topoisomerase Ⅱ activities in AML blasts and their correlation with celluar sensitivity to anthracyclines and epipodophyllotoxines. Leukemia. 1996;10:sup3 S46-S49．
32． Beck J, Niethammer D, et al. MDR1, MRP, Topoisomerase Ⅱ alpha/beta and cyclin gene expression in acute and chronic leukemias. Leukemia. 1996;10:sup S39-S45．
33． Kaufmann SH, Karp JE, Jones RJ, et al. Topoisomerase Ⅱ levels and drug sensitivity in adult acute myelogenous leukemia.Blood. 1994;83(2) :517-30．
34． Tsujimoto Y, Gossman J, Juffe E. et al. Involvement of the bcl-2 gene in human follicular lymphoma. Science. 1985,228:1140-1142．
35． Reed JC. Bcl-2 and the regulation of programmed cell death. J Cell Biol. 1994,124:1-3．
36． 郭晓玲，蔡作仁等．急性白血病患者bcl-2，bax基因表达与预后和耐 药的关系。中华血液学杂志．2001；22：39-41．
37． Salomons GS, Brady HJ, Verwijs-Janssen M,. et al. The Bax alpha: Bcl-2 ratio modulates the response to dexamethasone in leukemia cells and is highly variable in childhood acute leukaemia. Int J Cancer. 1997,71:959-965．
2． Fojo AT. Whang-Peng J, Gottesmann MM, Pastan I. Amplification of DNA sequences in human multidrug-resistance KB carcinoma cells. Proc Natl Acad Sci. USA. 1985;82:7661-7665．
3． Cole SPC. Bhardwaj G, Gerlach JH, et al. Overexpression of a transporter gene in a multidrug-resistance human lung cerner cell line. Science. 1992,258:1050-1664．
4． Scheper RJ, Broxterman HJ, Scheffer GL, et al. Overexpression of a M(r) 110,000 vesicular protein in non-P-glycoprotein-mediated multidrug resistance. Cancer Res. 1993;53:1475-1479．
5． Doyle L A, Yang Werdong, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Prol Natl Acad Sci. USA. 1998;95:15665-15670．
6． Lorico A, Rappa G, Srimatkandada S, et al. Increased rate of adenosine triphosphate-dependent etoposide (VP-16) efflux in a murine leukemia cell line overexpressing the multidrug resistomce-associated protein (MRP) gene. Cancer Res. 1995;55:4352-4360．
7． Patskovsky YY, Patskovska LN, Listowsky I, et al. Functions of His 107 in the catalytic mechanism of human glutathione s-transferase hGSTMla-la. Biochemistry. 1999;38:1193-1202．
8． Agular-Santelises M, Rottenberg ME, et al. Bcl-2, Bax and P53 expression in B-CLL in relation to in vitro survival and clinical progression. Int J Cancer. 1996;69:114-119．
9． 上海市白血病协作组，急性白血病患者P-gp，mdr-1，MRP和Topo II表达及其与预后关系的研究。中华血液学杂志，2001；22(2) ：90-93．
10． Arceci RJ. Clinical significance of P-glycoprotein in multidrug resistance malignancies. Blood. 1993;81:2215-2222．
11． Nooter K, Herweijer H. Multidrug resistance (mdr) genes in human cancer. Br J Cancer. 1991;63:663-669．
12． Cole SPC, Bhardwaj G, Gerlach JH. et al. Overexpression of a transporter gene in a multidrug-resistance human lung cancer cell line Science. 1992;258:1650-1664．
13． Nooter K, Burger H, Stoter G. Multidrug resistance-associated protein (MRP) in haematological malignancies. Leuk lymphoma. 1995;20:381-387．
14． Zhou DC, Zittoun R, Marie JP. Expression of the multidrug resistance-associated protein (MRP) and multidrug resistance (mdr-1) genes in acute myeloid leukemia. Leukemia. 1995;9:1661-1666．
15． Erasmus Schneider, Kenneth H, et al. Increased expression of the
multidrng resistance-associated protein gene in relapsed acute leukemia. Blood. 1995; 85: 186-193．
16． Herman Burger, Kees Nooter, et al. Expression of the multidrug resistance-associated protein (MRP) in acute and chronic leukemia. Leukemia. 1994;8:990-997．
17． Hart SM, GaneShaguru K, et al. Expression of the multidrug resistance-associated protein (MRP) in acute leukemia. Leukemia. 1994;8:2163-2168．
18． Hart SM, Ganeshaguru K, Scheper RJ, et al. Expression of the human major vault protein LRP in acute myeloid leukemia. Exp Hematol. 1997;25:1227-1232．
19． Rocchi E, Khodjakov A, Volk EL. et al. The product of the ABC half-transporter gene ABCG2(BCRP/MXR/ABCR) is expressed in the plasma membrane. Biochem Biophys Res commum. 2000;27 1:42-46．
20． Ross DD, Karp JE, Chen TT, et al. Expression of breast cancer resistance protein in blast cells from patients with acute leukemia. Blood. 2000,96:365-368．
21． 郑德先，吴克复，褚建新等．现代实验血液学研究方法与技术。 北京医科大学、中国协和医院大学联合出版社．1999． 4，第十版
22． Morrow CS, Cowan KH, Goldsmith ME, et al. Structure of the human genomic glutathione s-transferase-pi gene. Gene. 1989;75:3-11．
23． Tiirikainen MI, Elonen E, Syrjala MT, et al. Flow cytometric analysis
of glutathione S-transferase π in acute leukemia. Leukemia. 1994;8:978-984．
24． Sinha BK, Katki AG, Batist G, et al. Differential formation of hydroxyl radicals by adriamycin in sensitive and resistant MCF-7 human breast tumor cells implication for the mechanism of action. Biochemistry. 1987;26:3776-3781．
25． Litwack G, Kettever B, Arias LM, et al. Ligandin: A hepatic protein which binds steroids, bilirubin, carcinogens and a number of exogenous organic anions. Nature. 1997;234:466-467．
26． Massaad-Massade L, Ribrag V, Marie JP, et al. Glutathione system, topoisomerase Ⅱ level and multidrug resistance phenotype in acute myelogenous leukemia before treatment and at relapse. Anticancer Res. 1997;17:4647-4651．
27． Den Boer ML, Pieters R, et al. Different expression of glutathione S-transferase a , μ and π in childhood acute lymphoblastic and myeloid leukemia. Br J Haematol. 1999,104:321-327．
28． Rosario LA, O' Brien ML, Henderson CJ, et al. Cellular response to a glutathione s-transferase P1-1 activated prodrug. Mol Pharmacol, 2000;58:167-174．
29． Withoff S, Jong De S, Vries De EG, et al. Humanal DNA Topoisomerase Ⅱ: Biochemistry and role in chemotherapy resistance. Anticancer Res. 1996;16:1867-1870．
30． Beck J, Handgretinger R, Klingebiel T, et al. Expression of PKC isozyme and MDR-associated genes in primary and relapsed state. AML. Leukemia. 1996; 10:426-433．
31． Gieseler F, Glasmacher A, Kampfe D, et al. Topoisomerase Ⅱ activities in AML blasts and their correlation with celluar sensitivity to anthracyclines and epipodophyllotoxines. Leukemia. 1996;10:sup3 S46-S49．
32． Beck J, Niethammer D, et al. MDR1, MRP, Topoisomerase Ⅱ alpha/beta and cyclin gene expression in acute and chronic leukemias. Leukemia. 1996;10:sup S39-S45．
33． Kaufmann SH, Karp JE, Jones RJ, et al. Topoisomerase Ⅱ levels and drug sensitivity in adult acute myelogenous leukemia.Blood. 1994;83(2) :517-30．
34． Tsujimoto Y, Gossman J, Juffe E. et al. Involvement of the bcl-2 gene in human follicular lymphoma. Science. 1985,228:1140-1142．
35． Reed JC. Bcl-2 and the regulation of programmed cell death. J Cell Biol. 1994,124:1-3．
36． 郭晓玲，蔡作仁等．急性白血病患者bcl-2，bax基因表达与预后和耐 药的关系。中华血液学杂志．2001；22：39-41．
37． Salomons GS, Brady HJ, Verwijs-Janssen M,. et al. The Bax alpha: Bcl-2 ratio modulates the response to dexamethasone in leukemia cells and is highly variable in childhood acute leukaemia. Int J Cancer. 1997,71:959-965．