白血病多药耐药相关基因的研究
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摘要
白血病是严重危害人类健康的常见恶性肿瘤之一。目前，化疗仍是治疗白血病
的主要手段。近20余年来，随着抗癌新药的发现、支持疗法的改善、造血干细胞移
植的开展等，白血病的疗效有了一定的提高。但是，多数白血病病人的治疗仍以失
败而告终。对化疗药物产生多药耐药性（MDR）是白血病治疗失败的主要原因。
MDR，也称交叉耐药性，是指肿瘤细胞对某一抗癌药物产生耐药性的同时，对
其它结构和功能不同的抗癌药物也产生耐药性。国内外学者对MDR的发生机制进
行了多年的研究，发现了不少机制：药物转运蛋白过度表达、细胞解毒功能增强、
药物的作用靶点改变、细胞DNA损伤修复能力增强、细胞抗凋亡机制的形成等。
随着研究的不断深入，人们发现MDR发生的机制相当复杂。可以说，在抗癌
药物作用于肿瘤细胞、最终导致肿瘤死亡的过程中，每一个环节的改变都可能使抗
癌药物的作用效果受到影响，导致耐药性的形成。肿瘤细胞多药耐药性的形成极少
由单一机制介导，而往往是多种因素共同作用的结果。另外，目前已知的多药耐药
机制还不能完全解释肿瘤的MDR现象，尚存在未知的机制。因此，尚待进一步研
究阐明。
识别、分离、鉴定耐药细胞和敏感细胞间差异表达的基因是阐明MDR发生机
制的重要手段。抑制性差减杂交（suppression subtractive hybridization，SSH）是一种
研究基因差异表达的新方法，它应用抑制性PCR原理，可选择性地扩增两组组织或
细胞样本中差异表达的基因片段，从而使差异表达基因得到有效的分离，具有简便
易行、灵敏度高、重复性好等优点。SSH已在肿瘤相关基因的分离和鉴定中取得了
成效。
耐药肿瘤细胞株的建立是研究MDR发生机制的重要途径。浙江大学肿瘤研究
所建立的两种耐药细胞株K562/DOX和K562/VCR，对多种化疗药都具有不同程度
的抗性，是典型的MDR细胞。以往的研究发现，mrl基因和Pgp在这两种细胞
中高表达，是这两株细胞产生MDR的主要机制，但P－gp的表达尚不能完全解释其
MDR现象。因此，对这两株耐药细胞的进一步研究不仅有助于阐明其MDR发生机
制，更重要的是可能为白血病MDR机制的研究提供新线索。
本课题先用SSH技术分离、克隆耐药白血病细胞株（K562／DOX）及敏感细胞
株（K562）之间差异表达的基因，然后对克隆到的一些基因作表达和功能方面的进
一步研究，以期发现白血病MD K发生的新机制。
第一部分
K562八 和K562细胞间差异表达基因的分离和克隆
浙江大学肿瘤研究所建立的多药耐药白血病细胞株K562／DOX对多种化疗药物
具有耐药性，是典型的MDR细胞株。以往的研究标明mdrl基因和P－gp在这株细胞
中高表达，是其MDR发生的主要机制，但不是唯一机制。
采用钻H技术，分离K562和K562／DOX细胞间差异表达（K562／DOX细胞高表达）
的基因片断，并通过T／A克隆方法将差减杂交产物转化大肠肝菌，共获得了200余
个克隆。对其中80个克隆进行扩增，提取质粒鉴定，发现73个克隆有插入片断。
通过用K562和K562／DOX细胞的CDNA为探针对这73个克隆进行斑点杂交筛选，证
实其中14个克隆在K562／DOX细胞中高表达。对这14个克隆进行DNA序列测定，
并通过Iniamet与Genbank的已知基因序列比对，这14个克隆代表了门个己知基
因，分别是：
l．线粒体基因（Human lrTlll：oc，－．－－Mndria gCne，complete po）
2．NADH脱氢酶亚单位 2基因（Human NADH dehydrogenase subunit 2，NDZ）
3．电位依赖性阴离于通道3（VDAC3）基因（Human voltage－dependent aulon
channl）
4．S3核糖体蛋白基因（S3 ribosoma pfoteirsS3rp）
5．核糖体蛋白（L7a）基因（Human ibosomal ptein L7a）
6．铜／p超氧化物歧化酶侣OD）基因（Human Cu／Zn s－de dismutase）
7．干扰素诱导基因 l－su（Humanl－su gCne rom interferonlnducible gCne
— —ly）
8．血红蛋白 zeta基因（Human he。lobin zeta）
2
9．s期激酶激活剂基因（Human aCivato Ofs…ase inase）
10．白血病相关磷蛋白基因（Human fenk而a－associst6d…os…。以饥＊n）
（LAPI）（staben））
11．My023基因
以上基因产物中，除铜／＠超氧化物歧化酶（SOD）、线粒体与肿瘤耐药的
关系较明确外，其它基因在肿瘤耐药中的作用目前尚无定论。但分析结果表明，
这些基因可能与肿瘤MDR的发生有一定关系，可能代表了多药耐药的新机制。
这些基因有可能分别在MDR发生过程中的不同环节起作用，比如线粒体为Pgp
提供更多的ATP、Cu／ZnSOD阻止化疗药物对DNA的损伤作用、S3tl）加速损伤
DNA的修复等等，与P－gp一起最终导致了K562／DOX细胞MDR的发生。
第二部分
S3rp、NDZ和My023基因在不同白血病细胞株中表达的研究
在 11?
Even with the introduction of new anti-leukemic agents and approaches, most patients are still not cured. The occurrence of cross-resistance to structurally and functionally unrelated drugs, called multidrug resistsance(MDR), is a main cause of failure in the chemotherapy of leukemia. Several mechanisms of MDR have been identified, such as overexpression of drug transporter proteins, enhancement of detoxification enzymes and DNA repair enzymes, reduction of DNA topoisomerase , and decreased ability to initiate apoptosis. However, all the known alterations are not enough to explain fully the occurrence of MDR. In clinical situations, many patients have been reported to acquire resistance to chemotherapy without undergoing these changes, suggesting that other mechanisms could be involved.
Difference in gene expression are likely to explain the phenotypic difference between resistant tumor cells and sensitive tumor cells. Thus, it is an important approach to identify and characterize genes expressed exclusively or preferentially in resistant cancer cells. Suppression subtractive hybridization (SSH) is a new method to identify differentially expressed genes and proved to be a more rapid and reliable method than other techniques such as DDRT-PCR.
In this research, SSH was first performed to screen the differentially expressed genes between the MDR cell line K562/DOX and its parent cell line K562. Then the over-expressed genes in resistant leukemia cells were analyzed and further studied. Our goal is to find out novel mechanisms of MDR in leukemia.
Parti
Identification of differentially expressed genes between mult id rug resistant leukemic cell line K562/DOX and its parent cell line K562 using suppression subtractive hybridization
The multidrug resistanct leukemia cell line K562/DOX established at Cancer Institute of Zhejiang University is cross-resistant to quite a lot of antitumor drugs. Previous research has proved that overexpression of mdrlgene and P-glycoprotein is the main mechanism in the occurrence of MDR in this cell line but not the sole one.
First of all, we performed SSH to amplify the differentially expressed cDNA fragments between K562/DOX cell and K562 cell. After SSH, the subtracted and amplified products were cloned into pGEM-T Easy vector. Through T/A cloning, a subtracted library of about 200 clones in total was constructed. Thereafter, Dot Blot was carried out to further screen the subtracted library using cDNA probes from K562 cell and K562/DOX cell respectively. From 73 randomly selected clones, 14 clones were identified to be differentially expressed in MDR cell line K562/DOX. Sequence analysis and homology search of these clones indicated that these 14 clones represent 11 known genes:
Human mitochondria gene, complete genome,
Human NADH dehydrogenase subunit 2(ND2),
Human voltage-dependent anion channel 3 (VDAC3)
Human S3 ribosomal protein(S3rp),
Human ribosomal protein L7a,
Human Cu/Zn superoxide dismutase(Cu/Zn SOD),
Human l-8u gene from interferon-inducible gene family,
Human hemoglobin zeta,
Human activator of s phase kinase,
Human leukemia-associated phosphoprotein) (LAP 18)(Stathmin),
Human My023 protein(My023).
Among these gene products, increased expression of SOD has been shown to diminish
the cytotoxoic effects of certain chemotherapeutic agents. Enhancement of mitochondria energy-yielding pathways in drug resistant cancer cell has also been observed. However, so far the relationship between the other genes and the occurrence of multidrug resistance in cancer hasn't been reported. Thus, these genes may indicate novel mechanisms of MDR in leukemia.
Part II Expression of S3rp, ND2 and My023 genes in leukemia cell lines
In this research, semi-quantitative reverse transcription -polymerase chain reaction (RT-PCR), nucleic acid in situ hybridization and Northern Blot were used to study differential expression of S3rp, ND2 and My023 genes between sensitive and resistant leukemia cell lines
Results from semi-quantitative RT-PCR showed th
的主要手段。近20余年来，随着抗癌新药的发现、支持疗法的改善、造血干细胞移
植的开展等，白血病的疗效有了一定的提高。但是，多数白血病病人的治疗仍以失
败而告终。对化疗药物产生多药耐药性（MDR）是白血病治疗失败的主要原因。
MDR，也称交叉耐药性，是指肿瘤细胞对某一抗癌药物产生耐药性的同时，对
其它结构和功能不同的抗癌药物也产生耐药性。国内外学者对MDR的发生机制进
行了多年的研究，发现了不少机制：药物转运蛋白过度表达、细胞解毒功能增强、
药物的作用靶点改变、细胞DNA损伤修复能力增强、细胞抗凋亡机制的形成等。
随着研究的不断深入，人们发现MDR发生的机制相当复杂。可以说，在抗癌
药物作用于肿瘤细胞、最终导致肿瘤死亡的过程中，每一个环节的改变都可能使抗
癌药物的作用效果受到影响，导致耐药性的形成。肿瘤细胞多药耐药性的形成极少
由单一机制介导，而往往是多种因素共同作用的结果。另外，目前已知的多药耐药
机制还不能完全解释肿瘤的MDR现象，尚存在未知的机制。因此，尚待进一步研
究阐明。
识别、分离、鉴定耐药细胞和敏感细胞间差异表达的基因是阐明MDR发生机
制的重要手段。抑制性差减杂交（suppression subtractive hybridization，SSH）是一种
研究基因差异表达的新方法，它应用抑制性PCR原理，可选择性地扩增两组组织或
细胞样本中差异表达的基因片段，从而使差异表达基因得到有效的分离，具有简便
易行、灵敏度高、重复性好等优点。SSH已在肿瘤相关基因的分离和鉴定中取得了
成效。
耐药肿瘤细胞株的建立是研究MDR发生机制的重要途径。浙江大学肿瘤研究
所建立的两种耐药细胞株K562/DOX和K562/VCR，对多种化疗药都具有不同程度
的抗性，是典型的MDR细胞。以往的研究发现，mrl基因和Pgp在这两种细胞
中高表达，是这两株细胞产生MDR的主要机制，但P－gp的表达尚不能完全解释其
MDR现象。因此，对这两株耐药细胞的进一步研究不仅有助于阐明其MDR发生机
制，更重要的是可能为白血病MDR机制的研究提供新线索。
本课题先用SSH技术分离、克隆耐药白血病细胞株（K562／DOX）及敏感细胞
株（K562）之间差异表达的基因，然后对克隆到的一些基因作表达和功能方面的进
一步研究，以期发现白血病MD K发生的新机制。
第一部分
K562八 和K562细胞间差异表达基因的分离和克隆
浙江大学肿瘤研究所建立的多药耐药白血病细胞株K562／DOX对多种化疗药物
具有耐药性，是典型的MDR细胞株。以往的研究标明mdrl基因和P－gp在这株细胞
中高表达，是其MDR发生的主要机制，但不是唯一机制。
采用钻H技术，分离K562和K562／DOX细胞间差异表达（K562／DOX细胞高表达）
的基因片断，并通过T／A克隆方法将差减杂交产物转化大肠肝菌，共获得了200余
个克隆。对其中80个克隆进行扩增，提取质粒鉴定，发现73个克隆有插入片断。
通过用K562和K562／DOX细胞的CDNA为探针对这73个克隆进行斑点杂交筛选，证
实其中14个克隆在K562／DOX细胞中高表达。对这14个克隆进行DNA序列测定，
并通过Iniamet与Genbank的已知基因序列比对，这14个克隆代表了门个己知基
因，分别是：
l．线粒体基因（Human lrTlll：oc，－．－－Mndria gCne，complete po）
2．NADH脱氢酶亚单位 2基因（Human NADH dehydrogenase subunit 2，NDZ）
3．电位依赖性阴离于通道3（VDAC3）基因（Human voltage－dependent aulon
channl）
4．S3核糖体蛋白基因（S3 ribosoma pfoteirsS3rp）
5．核糖体蛋白（L7a）基因（Human ibosomal ptein L7a）
6．铜／p超氧化物歧化酶侣OD）基因（Human Cu／Zn s－de dismutase）
7．干扰素诱导基因 l－su（Humanl－su gCne rom interferonlnducible gCne
— —ly）
8．血红蛋白 zeta基因（Human he。lobin zeta）
2
9．s期激酶激活剂基因（Human aCivato Ofs…ase inase）
10．白血病相关磷蛋白基因（Human fenk而a－associst6d…os…。以饥＊n）
（LAPI）（staben））
11．My023基因
以上基因产物中，除铜／＠超氧化物歧化酶（SOD）、线粒体与肿瘤耐药的
关系较明确外，其它基因在肿瘤耐药中的作用目前尚无定论。但分析结果表明，
这些基因可能与肿瘤MDR的发生有一定关系，可能代表了多药耐药的新机制。
这些基因有可能分别在MDR发生过程中的不同环节起作用，比如线粒体为Pgp
提供更多的ATP、Cu／ZnSOD阻止化疗药物对DNA的损伤作用、S3tl）加速损伤
DNA的修复等等，与P－gp一起最终导致了K562／DOX细胞MDR的发生。
第二部分
S3rp、NDZ和My023基因在不同白血病细胞株中表达的研究
在 11?
Even with the introduction of new anti-leukemic agents and approaches, most patients are still not cured. The occurrence of cross-resistance to structurally and functionally unrelated drugs, called multidrug resistsance(MDR), is a main cause of failure in the chemotherapy of leukemia. Several mechanisms of MDR have been identified, such as overexpression of drug transporter proteins, enhancement of detoxification enzymes and DNA repair enzymes, reduction of DNA topoisomerase , and decreased ability to initiate apoptosis. However, all the known alterations are not enough to explain fully the occurrence of MDR. In clinical situations, many patients have been reported to acquire resistance to chemotherapy without undergoing these changes, suggesting that other mechanisms could be involved.
Difference in gene expression are likely to explain the phenotypic difference between resistant tumor cells and sensitive tumor cells. Thus, it is an important approach to identify and characterize genes expressed exclusively or preferentially in resistant cancer cells. Suppression subtractive hybridization (SSH) is a new method to identify differentially expressed genes and proved to be a more rapid and reliable method than other techniques such as DDRT-PCR.
In this research, SSH was first performed to screen the differentially expressed genes between the MDR cell line K562/DOX and its parent cell line K562. Then the over-expressed genes in resistant leukemia cells were analyzed and further studied. Our goal is to find out novel mechanisms of MDR in leukemia.
Parti
Identification of differentially expressed genes between mult id rug resistant leukemic cell line K562/DOX and its parent cell line K562 using suppression subtractive hybridization
The multidrug resistanct leukemia cell line K562/DOX established at Cancer Institute of Zhejiang University is cross-resistant to quite a lot of antitumor drugs. Previous research has proved that overexpression of mdrlgene and P-glycoprotein is the main mechanism in the occurrence of MDR in this cell line but not the sole one.
First of all, we performed SSH to amplify the differentially expressed cDNA fragments between K562/DOX cell and K562 cell. After SSH, the subtracted and amplified products were cloned into pGEM-T Easy vector. Through T/A cloning, a subtracted library of about 200 clones in total was constructed. Thereafter, Dot Blot was carried out to further screen the subtracted library using cDNA probes from K562 cell and K562/DOX cell respectively. From 73 randomly selected clones, 14 clones were identified to be differentially expressed in MDR cell line K562/DOX. Sequence analysis and homology search of these clones indicated that these 14 clones represent 11 known genes:
Human mitochondria gene, complete genome,
Human NADH dehydrogenase subunit 2(ND2),
Human voltage-dependent anion channel 3 (VDAC3)
Human S3 ribosomal protein(S3rp),
Human ribosomal protein L7a,
Human Cu/Zn superoxide dismutase(Cu/Zn SOD),
Human l-8u gene from interferon-inducible gene family,
Human hemoglobin zeta,
Human activator of s phase kinase,
Human leukemia-associated phosphoprotein) (LAP 18)(Stathmin),
Human My023 protein(My023).
Among these gene products, increased expression of SOD has been shown to diminish
the cytotoxoic effects of certain chemotherapeutic agents. Enhancement of mitochondria energy-yielding pathways in drug resistant cancer cell has also been observed. However, so far the relationship between the other genes and the occurrence of multidrug resistance in cancer hasn't been reported. Thus, these genes may indicate novel mechanisms of MDR in leukemia.
Part II Expression of S3rp, ND2 and My023 genes in leukemia cell lines
In this research, semi-quantitative reverse transcription -polymerase chain reaction (RT-PCR), nucleic acid in situ hybridization and Northern Blot were used to study differential expression of S3rp, ND2 and My023 genes between sensitive and resistant leukemia cell lines
Results from semi-quantitative RT-PCR showed th
引文
1 陈灏珠主编．内科学《高等医学院校教材》，1997；第四版：P570.
2 Arceci RJ. Can multidrug resistance mechanisms be modified? Br J Haemat, 2000; 110:285
3 Simon SM and Schindler M. Cell biologic mechanisms of multidrug resistance in tumors. Proc Natl Acad Sci, 1994,91:3497.
4 Mckenna SL. Multidrug ersistance in leukemia. Br J Haematol, 1997; 96: 659
5 Juliano RL, Ling V.A surface glycoprotein modulating drug permeability in Chinese hamster ovary mutants. Biochim Biochim Acta 1976;455:152-62.
6 Cole SP, Bhardwaj G, Gerlach JH, et al. Overexpression of a transport gene in a multidrug-resistant human lung cancer cell line. Science 1992;258:1650-4.
7 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;53:1475-9.
8 Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad USA 1998;95:15665-70
9 den Boer ML, Pieters R, Kazemier KM, et al. Different expression of glutathion S-transferase alpha, mu and pi in child acute lymphoblastic and myeloid leukemia, Br.J Haematol, 1999; 104:321
10 Kaufmann SH, Karp JE, Jones RJ, et al. TopoisomeraseⅡ Levels and drug sensitivity in adult acute myelogenous leukemia. Blood, 1994,83:517
11 Kim R, Beck WT. Differences between drug-sensitive and-resistant human leukemic CEM cells in c-jun expression, AP-1 DNA-binding activity, and formation of Jun/Fos family dimmers,and their association with internucleosomal DNA ladders after treatment with VM-26.Cancer Res, 1994;54:4958
12 Minn AJ,Rudin CM, Boise LH, et al. Expression of bcl-xL can confer a multidrug resistance phenotype. Blood, 1995;86:1903
13 Kim Sun-Hee, Um Jee-Hyun, Kim Dong-Won, et al. Potentiation of chemosensitivity in multidrug-resistant human leukemia CEM cells by inhibition of DNA-dependent protein kinase using wortmannin. Leukemia Res, 2000,24:917
14 Silberstein, Philip McGlave. Hematology:Basic Principles and Practice,3th Edition,P888
15 Diatchenko L, Lau YC, Campbell AP, et al. Suppression subtractive hybridization :a method for generating differentially regulated or tissure-speeific eDNA probes and libraries. Proc Natl Aead Sci USA, 1996,93:6025-6030
16 Diatchenko L, Lukyanov S, Lau YF, et al. Suppression subtractive hybridization:a versatile method for identifying differentially expressed genes. Methods Enzymol, 1999,303:349-380
17 胡汛，陈万源，蒽环类药物在多药抗药细胞株K562r细胞内的积聚、滞留与外排．中国药理学报，1994；15：275
18 胡汛，潘镪荣，郑树．P-糖蛋白过表达不能完全解释MDR细胞株K562／Dox的抗药性．中华肿瘤杂志，1995；17：85
2 Arceci RJ. Can multidrug resistance mechanisms be modified? Br J Haemat, 2000; 110:285
3 Simon SM and Schindler M. Cell biologic mechanisms of multidrug resistance in tumors. Proc Natl Acad Sci, 1994,91:3497.
4 Mckenna SL. Multidrug ersistance in leukemia. Br J Haematol, 1997; 96: 659
5 Juliano RL, Ling V.A surface glycoprotein modulating drug permeability in Chinese hamster ovary mutants. Biochim Biochim Acta 1976;455:152-62.
6 Cole SP, Bhardwaj G, Gerlach JH, et al. Overexpression of a transport gene in a multidrug-resistant human lung cancer cell line. Science 1992;258:1650-4.
7 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;53:1475-9.
8 Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad USA 1998;95:15665-70
9 den Boer ML, Pieters R, Kazemier KM, et al. Different expression of glutathion S-transferase alpha, mu and pi in child acute lymphoblastic and myeloid leukemia, Br.J Haematol, 1999; 104:321
10 Kaufmann SH, Karp JE, Jones RJ, et al. TopoisomeraseⅡ Levels and drug sensitivity in adult acute myelogenous leukemia. Blood, 1994,83:517
11 Kim R, Beck WT. Differences between drug-sensitive and-resistant human leukemic CEM cells in c-jun expression, AP-1 DNA-binding activity, and formation of Jun/Fos family dimmers,and their association with internucleosomal DNA ladders after treatment with VM-26.Cancer Res, 1994;54:4958
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