GST-Pi和P-gp在急性白血病中的表达及临床意义
摘要
目的
多药耐药(multi-drug resistance，MDR)是导致白血病化疗失败的主要原因。MDR是指肿瘤细胞一旦对某种药物产生耐受，对其它结构、作用机制不同的化疗药物也交叉耐受。其产生主要有以下几方面原因：①以细胞膜上的能量依赖式外排泵介导的MDR如MDR1、MRP(多药耐药蛋白)；②以胞浆中酶介导的MDR如GSTs(谷胱甘肽S-转移酶)；③以细胞核核孔蛋白介导的MDR如LRP(肺耐药相关蛋白)；④以细胞核内酶介导的MDR如TOPOⅡ(拓扑异构酶Ⅱ)。其中MDR1是较为经典的耐药途径，对其机理研究得也较为透彻。但对非经典耐药途径如GSTs、TOPOⅡ等研究的尚少，尤其是GSTs与耐药的关系尚不很明确。
GSTs是一组与机体解毒作用有关的酶类，根据其在细胞内定位的不同，一般可分为胞浆α、μ、π(Pi)、θ及膜结合微粒体5种类型，其中，GST-Pi与恶性肿瘤关系最密切，约占其总数的90％。目前有关GST-Pi在急性白血病中的表达及与预后的关系尚有争议，GST-Pi、P-gp在白血病中表达的相关性报道不一。为了进一步明确GST-Pi与急性白血病(AL)多药耐药的关系及与P-gp表达的相关性，本实验采用免疫组化方法检测160例AL患者GST-Pi及P-gp的表达，同时应用Habig等的方法检测其中25例患者GST-Pi的活性。结果表明，复发/难治组GST-Pi和P-gp的表达明显高于初治/敏感组，二者在复发/难治组中的表达密切相关；P-gp与GST-Pi共阳性的患者预后极差，二者是影响急性白血病患者预后的重要指标；GST-Pi表达与活性高度正相关。
方法
一、细胞培养
卵巢癌细胞株OVCAR3和白血病细胞株K-562/D及K-562/S按本实验室常规培养，所有实验均采用对数生长期细胞。OVCAR3和K-562/D细胞分别作为GST-Pi和P-gp表达的阳性对照，K-562/S作为P-gp表达的
阴性对照。
二、临床资料
AL患者160例，为我院1998年4月一2003年7月住院的患者，男95
例，女55例，中位年龄41岁，均符合FAB诊断标准。其中急性髓细胞白血
病(AML)104例，急性淋巴细胞白血病(ALL)50例。按治疗情况分为初治
/敏感组92例，复发/难治组68例。对照组取自同期在本院就诊的10例良
性血液病患者。按标准的诱导缓解方案进行正规化疗，复发/难治AML组
选用MA、IA方案，复发/难治LL组选用大剂量MTX或Ara一C方案。
三、单个核细胞分离
取肝素抗凝骨髓3一5血(幼稚细胞>70%)，用Ficon密度梯度离心
法分离单个核细胞。
四、GST一Pi表达的检测
应用免疫组化方法检测GST一Pi和P一gP的表达，以P一gP和GST一Pi表达
阳性的幼稚细胞数分别>20%和25%分别判定P一gP和GST一Pi阳性。
五、GST一Pi活性的检测
用Habig等的方法测定GST一Pi活性。GST一Pi活性以每分钟每毫克胞
浆蛋白与利尿酸/谷胧甘肤共扼的卿d数来表示(runol/mi可mg)。
六、统计学处理
采用x，检验、t检验和直线相关分析。
结果
一、初治/敏感组与复发/难治组GST~Pi和P一gP表达的比较
GST一Pi和P一gP的总阳性率分别为40.6%(65/160)和53.1%(85/
100);GST一Pi在初治/敏感组与复发/难治组中的阳性率分别是24 .5%(23/
92)及62.4%(42/68);P-gP在初治/敏感组与复发/难治组中的阳性率分
别是36.4%(34/92)及75.3%(51/68)，P值均<0.05，差异均有显著性意
义。
二、GST-pi和P一gP的表达关系
对160例白血病患者仪汀.乃和P一的表达进行直线相关分析，结果发
现初治/敏感组相关系数r=0.21，P>0.05;复发/难治组相关系数r二
0 .75，P<0.05。
三、GST一R和P一gP的表达与临床疗效关系的比较
全部患者均经两个疗程标准化疗后评价疗效，以完全缓解(CR)为有
效。CST一Pi表达阳性与阴性组的完全缓解率分别为26.2%(17/75)和
78 .9%(75/85)，P<0.05;P-gP表达阳性与阴性组的完全缓解率分别为
29 .1%(25/85)和89.3%(67刀5)，P值<0.05，二者均有显著性差异。P-
gP与GST-Pi共阳性者与共阴性者的CR率分别为12.7%(7/55)和珍2.3%
(印/65)，P<0.05，差别有显著性意义。
四、GST一Pi活性检测结果
正常对照组CST一Pi活性为10.12土5.41(nmol/而。/毗)，AML组g汀.
Pi活性为38.61土10.巧(恤d/面n/mg)，川比组CS卜R活性为44.10土
11 .62(nznol/min/mg)，AML组与ALL组CST书活性均明显高于正常对照
组(P值均<0 .05);AML组与ALL组CST‘R活性无显著差异，P>0.05。
五、 GST一Pi表达与活性的关系
经线性相关分析发现，GST一Pi表达与活性呈高度正相关，r=0.71，P
<0.05，其中有两例患者CST一R高表达低活性。
结论
1 .GST一Pi和P一gP在AL复发/难治组中的表达明显高于初治组，二者
在AL复发/难治组中的表达密切相关。
2 .GST一Pi和P一gP共阳性的AL患者预后极差，二者是影响AL预后的
重要指标。
3 .GST一Pi在AL中的表达与活性呈高度正相关。
Objective
Multi - drug resistance ( MDR) is a main reason to cause leukemia chemotherapy failed . The MDR is characterized by cross - resistance to avariety of drugs with different structure and function ,Its creation has primarily below several aspects reason : The (1) depends on the energy of the cell membrane efflux pump , such as P-gp, MRP(multi -drug resistance relevant protein) ; The (2) straches with the cytosolic enzymes such as GSTs(glutathion -S - thansferas-es) ; The (3) lies the MDR ,such as LRP(lung resistant relevant protein) , that lead with the cell nucleus spot protein ; The (4) lies the MDR , such as TOPO II (topoisomerase II) that lead with the enzymes inside the cell nucleus. Among them , P-gp is the more classical one ,as to its mechanism study too more very clear. But to not the classical one ,such as GSTs etc. investigation is still little.
GSTs may contribute to the cellular resistance by detoxifying cytostatic a-gents . According to its in the already decided in cell of different, GSTs generally can be divided into a , u, , IT ( PI) , 6 and membrane combinating micro-somal 5 types, among them, GST - Pi is the most close with malignant tumor relation, inviting to account for 90% of its total amount . The drug resistant function of GST-Pi is mainly caused by its detoxifying , its mechanisms thinks now below several aspects : (I) GSTs may catalyse the conjugation of reduced glutathione (GSH) to electrophilic molecules , such as the alkylating agents , which may enhance the solubility and excretion of the drug ; (2) GSTs may also detoxify the radicals formed by drug , e. g. the oxygen and hydroxyl radicals generated by anthracyclines ; (3) GSTs can also sequester drugs by direct binding , e. g. of alkylation agents and steroids ; (4) GSTs still has the activity of GSH peroxidase . In this paper , to understand the relation between GST-Pi and MDR
in AL ,we analyzed the expression of GST-Pi and P-gp in 160 cases AL by
immunohistochemistry , at the same time , the GST-Pi activity in 25 cases were also examined by Habig etc methods . The results showed that the expression of GST-Pi and P-gp in relapsed / refractory groups were higher than in primary / sensitive groups , there were close relation between the expression of GST-Pi and P-gp in relapsed / refractory groups , GST-Pi and P-gp were the important factors in the effect of prognosis in AL; the co - expression of GST-Pi and P-gp lead to a poorer prognosis . there were a higher relation between the expression and activity of GST-Pi.
Methods
1. cell culture : Drug resistant cell lines OVCAR3 and K - 562 were cultured routinely in our laboratory . Exponentially growing cells were used for all experiments . The tow cell lines were used as positive controls for GST-Pi and P-gp , respectively.
2. Patient samples ; 160 cases AL patients ,95 were male , 55 were female average age was 41 , all cases were confirmed according to FAB diagnostic standard . Among them , there were 104 AML cases and 50 ALL cases . According to the outcome , 92 primary / sensitive cases and 68 relapsed / refractory cases . 10 of non - tumor cases were used as normal controls . Standard induced remission chemotherapy were used.
3. Isolation of mononuclear cells ; 3 - 5 ml heparipheral bone marrow fluid (blasts >70% ) were collected , mononuclear cells were isolated with Ficoll density gradient centrifugation.
4. Detection of GST-Pi and P-gp expression : GST-Pi and P-gp expression were monitored by immunohistochemistry and were judged positive ceil > 25% and 20% , respectively.
5. Detection of GST-Pi activity :
GST-Pi activity was monitored by Habig etc. methods.
6. Statistical methods:
X test ,t test and liner correlation analysis were used.
Results
1. Expression of GST-Pi and P-gp in primary/sensitive groups and relapsed/refractory groups
The total positive rates of GST-Pi and P-gp were 40. 6% and 53. 1% , respectively ; the positive rates of GST-Pi in primary/sensitive groups and relapsed/refractory groups were 24. 5% and
多药耐药(multi-drug resistance，MDR)是导致白血病化疗失败的主要原因。MDR是指肿瘤细胞一旦对某种药物产生耐受，对其它结构、作用机制不同的化疗药物也交叉耐受。其产生主要有以下几方面原因：①以细胞膜上的能量依赖式外排泵介导的MDR如MDR1、MRP(多药耐药蛋白)；②以胞浆中酶介导的MDR如GSTs(谷胱甘肽S-转移酶)；③以细胞核核孔蛋白介导的MDR如LRP(肺耐药相关蛋白)；④以细胞核内酶介导的MDR如TOPOⅡ(拓扑异构酶Ⅱ)。其中MDR1是较为经典的耐药途径，对其机理研究得也较为透彻。但对非经典耐药途径如GSTs、TOPOⅡ等研究的尚少，尤其是GSTs与耐药的关系尚不很明确。
GSTs是一组与机体解毒作用有关的酶类，根据其在细胞内定位的不同，一般可分为胞浆α、μ、π(Pi)、θ及膜结合微粒体5种类型，其中，GST-Pi与恶性肿瘤关系最密切，约占其总数的90％。目前有关GST-Pi在急性白血病中的表达及与预后的关系尚有争议，GST-Pi、P-gp在白血病中表达的相关性报道不一。为了进一步明确GST-Pi与急性白血病(AL)多药耐药的关系及与P-gp表达的相关性，本实验采用免疫组化方法检测160例AL患者GST-Pi及P-gp的表达，同时应用Habig等的方法检测其中25例患者GST-Pi的活性。结果表明，复发/难治组GST-Pi和P-gp的表达明显高于初治/敏感组，二者在复发/难治组中的表达密切相关；P-gp与GST-Pi共阳性的患者预后极差，二者是影响急性白血病患者预后的重要指标；GST-Pi表达与活性高度正相关。
方法
一、细胞培养
卵巢癌细胞株OVCAR3和白血病细胞株K-562/D及K-562/S按本实验室常规培养，所有实验均采用对数生长期细胞。OVCAR3和K-562/D细胞分别作为GST-Pi和P-gp表达的阳性对照，K-562/S作为P-gp表达的
阴性对照。
二、临床资料
AL患者160例，为我院1998年4月一2003年7月住院的患者，男95
例，女55例，中位年龄41岁，均符合FAB诊断标准。其中急性髓细胞白血
病(AML)104例，急性淋巴细胞白血病(ALL)50例。按治疗情况分为初治
/敏感组92例，复发/难治组68例。对照组取自同期在本院就诊的10例良
性血液病患者。按标准的诱导缓解方案进行正规化疗，复发/难治AML组
选用MA、IA方案，复发/难治LL组选用大剂量MTX或Ara一C方案。
三、单个核细胞分离
取肝素抗凝骨髓3一5血(幼稚细胞>70%)，用Ficon密度梯度离心
法分离单个核细胞。
四、GST一Pi表达的检测
应用免疫组化方法检测GST一Pi和P一gP的表达，以P一gP和GST一Pi表达
阳性的幼稚细胞数分别>20%和25%分别判定P一gP和GST一Pi阳性。
五、GST一Pi活性的检测
用Habig等的方法测定GST一Pi活性。GST一Pi活性以每分钟每毫克胞
浆蛋白与利尿酸/谷胧甘肤共扼的卿d数来表示(runol/mi可mg)。
六、统计学处理
采用x，检验、t检验和直线相关分析。
结果
一、初治/敏感组与复发/难治组GST~Pi和P一gP表达的比较
GST一Pi和P一gP的总阳性率分别为40.6%(65/160)和53.1%(85/
100);GST一Pi在初治/敏感组与复发/难治组中的阳性率分别是24 .5%(23/
92)及62.4%(42/68);P-gP在初治/敏感组与复发/难治组中的阳性率分
别是36.4%(34/92)及75.3%(51/68)，P值均<0.05，差异均有显著性意
义。
二、GST-pi和P一gP的表达关系
对160例白血病患者仪汀.乃和P一的表达进行直线相关分析，结果发
现初治/敏感组相关系数r=0.21，P>0.05;复发/难治组相关系数r二
0 .75，P<0.05。
三、GST一R和P一gP的表达与临床疗效关系的比较
全部患者均经两个疗程标准化疗后评价疗效，以完全缓解(CR)为有
效。CST一Pi表达阳性与阴性组的完全缓解率分别为26.2%(17/75)和
78 .9%(75/85)，P<0.05;P-gP表达阳性与阴性组的完全缓解率分别为
29 .1%(25/85)和89.3%(67刀5)，P值<0.05，二者均有显著性差异。P-
gP与GST-Pi共阳性者与共阴性者的CR率分别为12.7%(7/55)和珍2.3%
(印/65)，P<0.05，差别有显著性意义。
四、GST一Pi活性检测结果
正常对照组CST一Pi活性为10.12土5.41(nmol/而。/毗)，AML组g汀.
Pi活性为38.61土10.巧(恤d/面n/mg)，川比组CS卜R活性为44.10土
11 .62(nznol/min/mg)，AML组与ALL组CST书活性均明显高于正常对照
组(P值均<0 .05);AML组与ALL组CST‘R活性无显著差异，P>0.05。
五、 GST一Pi表达与活性的关系
经线性相关分析发现，GST一Pi表达与活性呈高度正相关，r=0.71，P
<0.05，其中有两例患者CST一R高表达低活性。
结论
1 .GST一Pi和P一gP在AL复发/难治组中的表达明显高于初治组，二者
在AL复发/难治组中的表达密切相关。
2 .GST一Pi和P一gP共阳性的AL患者预后极差，二者是影响AL预后的
重要指标。
3 .GST一Pi在AL中的表达与活性呈高度正相关。
Objective
Multi - drug resistance ( MDR) is a main reason to cause leukemia chemotherapy failed . The MDR is characterized by cross - resistance to avariety of drugs with different structure and function ,Its creation has primarily below several aspects reason : The (1) depends on the energy of the cell membrane efflux pump , such as P-gp, MRP(multi -drug resistance relevant protein) ; The (2) straches with the cytosolic enzymes such as GSTs(glutathion -S - thansferas-es) ; The (3) lies the MDR ,such as LRP(lung resistant relevant protein) , that lead with the cell nucleus spot protein ; The (4) lies the MDR , such as TOPO II (topoisomerase II) that lead with the enzymes inside the cell nucleus. Among them , P-gp is the more classical one ,as to its mechanism study too more very clear. But to not the classical one ,such as GSTs etc. investigation is still little.
GSTs may contribute to the cellular resistance by detoxifying cytostatic a-gents . According to its in the already decided in cell of different, GSTs generally can be divided into a , u, , IT ( PI) , 6 and membrane combinating micro-somal 5 types, among them, GST - Pi is the most close with malignant tumor relation, inviting to account for 90% of its total amount . The drug resistant function of GST-Pi is mainly caused by its detoxifying , its mechanisms thinks now below several aspects : (I) GSTs may catalyse the conjugation of reduced glutathione (GSH) to electrophilic molecules , such as the alkylating agents , which may enhance the solubility and excretion of the drug ; (2) GSTs may also detoxify the radicals formed by drug , e. g. the oxygen and hydroxyl radicals generated by anthracyclines ; (3) GSTs can also sequester drugs by direct binding , e. g. of alkylation agents and steroids ; (4) GSTs still has the activity of GSH peroxidase . In this paper , to understand the relation between GST-Pi and MDR
in AL ,we analyzed the expression of GST-Pi and P-gp in 160 cases AL by
immunohistochemistry , at the same time , the GST-Pi activity in 25 cases were also examined by Habig etc methods . The results showed that the expression of GST-Pi and P-gp in relapsed / refractory groups were higher than in primary / sensitive groups , there were close relation between the expression of GST-Pi and P-gp in relapsed / refractory groups , GST-Pi and P-gp were the important factors in the effect of prognosis in AL; the co - expression of GST-Pi and P-gp lead to a poorer prognosis . there were a higher relation between the expression and activity of GST-Pi.
Methods
1. cell culture : Drug resistant cell lines OVCAR3 and K - 562 were cultured routinely in our laboratory . Exponentially growing cells were used for all experiments . The tow cell lines were used as positive controls for GST-Pi and P-gp , respectively.
2. Patient samples ; 160 cases AL patients ,95 were male , 55 were female average age was 41 , all cases were confirmed according to FAB diagnostic standard . Among them , there were 104 AML cases and 50 ALL cases . According to the outcome , 92 primary / sensitive cases and 68 relapsed / refractory cases . 10 of non - tumor cases were used as normal controls . Standard induced remission chemotherapy were used.
3. Isolation of mononuclear cells ; 3 - 5 ml heparipheral bone marrow fluid (blasts >70% ) were collected , mononuclear cells were isolated with Ficoll density gradient centrifugation.
4. Detection of GST-Pi and P-gp expression : GST-Pi and P-gp expression were monitored by immunohistochemistry and were judged positive ceil > 25% and 20% , respectively.
5. Detection of GST-Pi activity :
GST-Pi activity was monitored by Habig etc. methods.
6. Statistical methods:
X test ,t test and liner correlation analysis were used.
Results
1. Expression of GST-Pi and P-gp in primary/sensitive groups and relapsed/refractory groups
The total positive rates of GST-Pi and P-gp were 40. 6% and 53. 1% , respectively ; the positive rates of GST-Pi in primary/sensitive groups and relapsed/refractory groups were 24. 5% and
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20. Rosario LA, OBrien ML, Henderson CJ,et al: Cellular response to a glutathione S-transferase P1-1 activated predrug. Mol Pharmacol. 2000 Jul; 58(1):167-174.
21. Schipper DL, Wagenmans MJM, Van Haelst U, et al: Immunohistochemical determination of glutathione S-transferases in gastric carcinomas and in adjacent normal gastric epithelium. Anticancer Res. 1996,16:565-572.
22. Volm M. Overexpression of P-glycoprotein and ghtathion s-transferase-π in resistant non- small- cell lung carcinomas of smokers. Br J Cancer,
1991,64:700 - 704.
23. Ma K P, Daniela D S, Adriana F, et al. GST, π and P- 170 co- expression in multiple myeloma. Br J heamatol. 1995,90:393 -401.
24. Holmes, J., Wareing, C., Jacobs, A., et al: Glutathione -s- transferase pi expression in leukemia: a comparative analysis with mdr-1 data. British Journal of Cancer. 1990,62:209-212.
25. Russo, D., Marie, J. P., Zhou, D.C. et al: Coexpression of anionic glutathione-S-transferase (GST-Pi) and multidrug resistance (mdrl) genes in acute myeloid and lymphoid leukemias. Leukemia. 1994,8:881-884.
26. Linsenmeyer, M.E., Jefferson, S., Wolf M., et al : Levels of expression of the mdrl gene and glutathione S-transferase genes 2 and 3 and response to chemotherapy in multiple myeloma. British Journal of Cancer. 1992,65: 471-475.
27．温宏升，夏薇，盛瑞兰，等．急性髓细胞白血病谷胱甘肽-S-转移酶π表达及其临床意义．中国实验血液学杂志．1999，7(20)：156-158．
28．张春青，徐从高，张锑，等急性白血病P170和GSTπ的表达及临床意义．临床肿瘤学杂志，1997，2(1)：19-21．
29. Ikeguchi, M., Teeter, L. D., Eckersberg, T., et al: Structural and functional analyses of the promoter of the routine multidrug resistance gene mdr3/mdr1 a reveal a negative dement containing the AP-1 binding site. DNA and Cell Biology. 1991,10:639 -649.
30. Bergelson, S., Pinkus, R., Danlel, V.: Intracellular glutathione levels regulate Fos/Jun induction and activation of glutathione S-transferase gene expression. Cancer Research. 1994, 54:36-40.
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32. Koomagi R,Stammler C, Manegold C, et al: Expression of resistance-related protein in tumoral and peritumoral tissues of patients with lung cancer. Cancer Lett. 1996,110:129.
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15. Tiirikainen MI, Elonen E, Syrjala MT,et al:. Flow cytometric analysis of ghtathione-S-transferase-pi in acute leukemia. Leukemia. 1994 Jun;8 (6):978-84.
16. Koberda J and Hellman A: Glutathione S-transferase activity of leukemic cells as a prognostic factor for response to chemotherapy in acute leukemias. Med Oncol Tumor Pharmacother. 1991,8:35-38.
17. Tidefelt U, Elmhom-Rosenborg A, Paul C, et al: Expression of glutathione S-transferase π as a predictor for treatment results at different stages of acute nonlymphoblastic leukemia. Cancer Res. 1992,52:3281-3285.
18. Joncourt F, Oberli-Schrammli AE, Stadler M, et al: Patterns of drug resistance parameters in adult leukemia. Leuk Lymphoma. 1995,17:101-109.
19. OBrien ML, Tew KD. Glutathione and related enzymes in multidrug resistance. Eur J cancer. 1996,32A : 967-978.
20. Rosario LA, OBrien ML, Henderson CJ,et al: Cellular response to a glutathione S-transferase P1-1 activated predrug. Mol Pharmacol. 2000 Jul; 58(1):167-174.
21. Schipper DL, Wagenmans MJM, Van Haelst U, et al: Immunohistochemical determination of glutathione S-transferases in gastric carcinomas and in adjacent normal gastric epithelium. Anticancer Res. 1996,16:565-572.
22. Volm M. Overexpression of P-glycoprotein and ghtathion s-transferase-π in resistant non- small- cell lung carcinomas of smokers. Br J Cancer,
1991,64:700 - 704.
23. Ma K P, Daniela D S, Adriana F, et al. GST, π and P- 170 co- expression in multiple myeloma. Br J heamatol. 1995,90:393 -401.
24. Holmes, J., Wareing, C., Jacobs, A., et al: Glutathione -s- transferase pi expression in leukemia: a comparative analysis with mdr-1 data. British Journal of Cancer. 1990,62:209-212.
25. Russo, D., Marie, J. P., Zhou, D.C. et al: Coexpression of anionic glutathione-S-transferase (GST-Pi) and multidrug resistance (mdrl) genes in acute myeloid and lymphoid leukemias. Leukemia. 1994,8:881-884.
26. Linsenmeyer, M.E., Jefferson, S., Wolf M., et al : Levels of expression of the mdrl gene and glutathione S-transferase genes 2 and 3 and response to chemotherapy in multiple myeloma. British Journal of Cancer. 1992,65: 471-475.
27．温宏升，夏薇，盛瑞兰，等．急性髓细胞白血病谷胱甘肽-S-转移酶π表达及其临床意义．中国实验血液学杂志．1999，7(20)：156-158．
28．张春青，徐从高，张锑，等急性白血病P170和GSTπ的表达及临床意义．临床肿瘤学杂志，1997，2(1)：19-21．
29. Ikeguchi, M., Teeter, L. D., Eckersberg, T., et al: Structural and functional analyses of the promoter of the routine multidrug resistance gene mdr3/mdr1 a reveal a negative dement containing the AP-1 binding site. DNA and Cell Biology. 1991,10:639 -649.
30. Bergelson, S., Pinkus, R., Danlel, V.: Intracellular glutathione levels regulate Fos/Jun induction and activation of glutathione S-transferase gene expression. Cancer Research. 1994, 54:36-40.
31. Moffat, G.J., Melaren, A.W., Wolf, C.R.: Involvement of Jun and Fos proteins in regulating transcriptional activation of the human pi class glutathione S-transferase gene in multidrug resistant MCF7 breast cancer cells. Journal of Biological Chemistry. 1995,269:16397 - 16402.
32. Koomagi R,Stammler C, Manegold C, et al: Expression of resistance-related protein in tumoral and peritumoral tissues of patients with lung cancer. Cancer Lett. 1996,110:129.