血清YB-1蛋白定量检测方法的建立及其作为肿瘤标志物的探讨
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摘要
目的：
1.建立基因工程制备YB-1蛋白（Y-box binding protein1，YB-1)系统，分离纯化YB-1蛋白，制备其多克隆抗体及单克隆抗体；
3.构建以化学发光免疫分析法（chemiluminescence immunoassay，CLIA）为基础的YB-1定量免疫学检测技术平台；
3. CLIA定量检测人血清YB-1，并初步探讨其作为常见恶性肿瘤辅助诊断标志物的临床意义。
方法：
1.采用RT-PCR技术，从肿瘤细胞总RNA中扩增YB-1编码序列，将其克隆到pGEX-6p-1原核表达载体，构建重组表达质粒pGEX/YB-1；
2. pGEX/YB-1转化大肠杆菌BL21（DE3），确定最佳的可溶性诱导表达条件，GST亲和层析与PSP柱上蛋白酶切纯化靶蛋白；
3.将纯化的YB-1免疫动物，制备其多克隆抗体；杂交瘤法制备YB-1单克隆抗体；进一步采用B细胞抗原表位预测法初步确定单抗识别的抗原表位；
4.用制备的YB-1多抗与单抗，建立血清YB-1定量化学发光免疫学检测方法（CLIA），并对建立的CLIA法进行方法学和检测性能评价；
5.以建立的CLIA定量测定健康人、良性与恶性肿瘤患者血清中YB-1蛋白水平，并初步评价其作为常见恶性肿瘤标志物的临床意义；
6.同时与甲胎蛋白的ROC曲线比较对原发性肝癌的诊断性能。
结果：
1.采用基因克隆技术成功构建了重组载体pGEX/YB-1；经表达、分离、纯化与蛋白酶切GST，获取重组YB-1蛋白；将其免疫动物制备了YB-1多抗；
2.建立了稳定分泌mAb且能特异识别YB-1的杂交瘤细胞株（1-D9、3-E8）；通过纯化制备了效价与亲和力较高的YB-1mAb；
3.杂交瘤细胞株1-D9和3-E8所分泌单抗的识别表位分别位于YB-1蛋白的（134-160aa）与（266-303aa）肽段；
4.建立了定量检测YB-1的CLIA检测方法；该法最低检测限为0.1μg/L，线性范围1.0-150.0μg/L，批内与批间变异系数分别为≤4.8和≤12.5%，平均回收率为100.6%，整个检测所需时间约90min；
5.初步确定了良性组（11.62±2.97μg/L）、恶性肿瘤组（18.69±6.11μg/L）以及健康组（10.79±2.39μg/L）血清YB-1水平；恶性肿瘤患者血清YB-1水平显著高于良性肿瘤患者及健康人（P<0.0001）；
6. ROC曲线显示，YB-1曲线下面积为0.896(P <0.0001)，在cut-off值≥13.45μg/L的条件下，血清YB-1辅助诊断恶性肿瘤的灵敏度为80%，特异性为81%；
7. ROC曲线分析表明，在诊断原发性肝癌上，YB-1的AUC为0.901（P <0.0001），在cut-off值＞14.69μg/L，血清YB-1辅助诊断原发性肝癌的灵敏度为84%，特异性为77%。而AFP的AUC为0.912（P <0.001），在cut-off值＞425.0μg/L，血清AFP辅助诊断原发性肝癌的灵敏度为78%，特异性为84%。
结论：
1.成功制备了重组YB-1蛋白及其多抗与单抗，并初步确定了单抗所识别的抗原表位；
2.成功建立了具较好检测性能的血清YB-1定量检测CLIA法；
3.血清YB-1水平可作为常见恶性肿瘤辅助诊断的标志物；
4.血清YB-1诊断原发性肝癌的效能与AFP接近；
5.该研究工作为进一步评价血清YB-1作为恶性肿瘤预后判断标志物的临床评价以及该法的临床推广应用提供了良好基础。
Objective:
1. Establishment of genetic engineering system to prepare recombinanthuman Y-box binding protein1(YB-1); and preparation of polyclonalantibody (pAb) and monoclonal antibody (mAb) against YB-1.
2. Development of a chemiluminescence immunoassay (CLIA) methodfor quantifying serum YB-1.
3. Quantitatifing serum YB-1with established CLIA, and investigatingthe clinical significance of serum YB-1as an aid-diagnostic biomarkerfor common malignancy tumors.
Methods:
1. YB-1coding sequence was obtained by RT-PCR using the total RNAof human renal cancer cell line786-0as template. The sequence wascloned into vector pGEX-6P-1and confirmed by double-enzymedigestion, and further identified by DNA sequencing.
2. For prokaryotic expression of YB-1, pGEX/YB-1was transformed into E. coli to express fusion protein GST-YB1. GST-affinitychromatography and chromatographic column protease digestion wereapplied to purify the recombinant YB-1protein.
3. PAb against human YB-1was raised by injecting a rabbitsubcutaneously four times at2-week intervals with2mg each time ofthe recombinant human YB-1. The IgG fraction of antiserum waspurified by protein A sepharose column chromatography. Anti-YB-1mAbs used in CLIA of human sera YB-1were prepared by hybridomatechnique; then B-lymphocytes epitope prediction were applied toidentify the epitopes recognition by YB-1mAbs.
4. The prepared pAb and mAb against YB-1were used to establish CLIAfor quantifying serum YB-1. Methodology evaluation was carried outto analysis the performance of CLIA.
5. The concentrations of serum YB-1in patients with malignant cancer(n=157), and benign tumor (n=30) and healthy individuals (n=100) ascontrol groups were determined by CLIA; and preliminary evaluationof clinical significance of serum YB-1as an aid-diagnostic tumormarker was performed.
6. Receiver operating characteristics (ROC) was used to compare thediagnostic performance of serum YB-1and alpha fetoprotein (AFP) fordiogmosis of hepatocellular carcinoma (HCC).
Results：
1. The vector pGEX/YB-1was successfully constructed and identified.The purified YB-1protein was prepared by means of prokaryoticexpression, GST-affinity chromatography and chromatographic columnprotease digestion.
2. YB-1pAb was obtained by immunizing rabbits.2hybridoma cell lines(1-D9,3-E8) stably secreting mAb against YB-1were obtained, thesemAbs could specifically recognize exogenous and endogenous YB-1.
3. The recognized epitope of the2mAbs might harbor in134-160aa and266-303aa of YB-1protein sequence.
4. CLIA was established based on double-antibody sandwich. Thecalibration curve of the CLIA had a coefficient of linear correlation r2>0.99(slope0.0702, intercept5.8112). The minimum detection limit was0.1μg/L; the linear range was from1.0to150.0μg/L; the intra-andinter-assay CV values were≤4.8and≤12.5%at10.0μg/L, respectively.The average recovery was100.6%(93.9%~109.0%). The total time ofthe assay was around90min.
5. With CLIA assay, the mean (SD) concentration of YB-1in100healthyserum samples was10.79±2.39μg/L; in30patients with benign tumorwas11.62±2.97μg/L; in157cancer patients was18.69±6.11μg/L,which was significantly higher than the values of healthy and benign tumor groups (P <0.0001).
6. ROC analysis demonstrated that the area under a ROC curve (AUC) forthe YB-1curve was0.896(P <0.0001). Serum YB-1had a sensitivityof80%(95%CI73%-86%), a specificity of81%(95%CI73%-87%)at the cut-off value of13.45μg/L for diagnosis cancer.
7. For diagnosis of HCC, the AUC for the YB-1curve was0.901(P <0.0001). Serum YB-1had a sensitivity of84%, a specificity of77%atthe cut-off value of14.69μg/L, while the AUC for the AFP curve was0.912(P <0.0001). AFP had a sensitivity of78%, a specificity of84%at the cut-off value of425.0μg/L.
Conclusions:
1. The recombinant YB-1protein and its pAbs and mAbs are successfullyprepared, and the recognized epitope of the two mAbs (1-D9,3-E8) isconfirmed.
2. A high-performance double antibody sandwich CLIA for thequantitative measurement of serum YB-1with the combination ofMAbs1-D9and Anti-YB-1PAbs has been developed.
3. Serum concentrations of YB-1might be used as a biomarker ofaided-diagnosis of common malignant tumors.
4. The diagnostic performance of serum YB-1is close to AFP for diagnosis of HCC.
5. Our primary exploratory study has suggested that quantifying serumYB-1, as an easily accessible circulating biomarker, could offer usefulaid-diagnostic information for patients with cancer; and this study laysfoundations for clinical application of the established CLIA in tumoraid-diagnosis and further evaluation of cancer prognosis.
1.建立基因工程制备YB-1蛋白（Y-box binding protein1，YB-1)系统，分离纯化YB-1蛋白，制备其多克隆抗体及单克隆抗体；
3.构建以化学发光免疫分析法（chemiluminescence immunoassay，CLIA）为基础的YB-1定量免疫学检测技术平台；
3. CLIA定量检测人血清YB-1，并初步探讨其作为常见恶性肿瘤辅助诊断标志物的临床意义。
方法：
1.采用RT-PCR技术，从肿瘤细胞总RNA中扩增YB-1编码序列，将其克隆到pGEX-6p-1原核表达载体，构建重组表达质粒pGEX/YB-1；
2. pGEX/YB-1转化大肠杆菌BL21（DE3），确定最佳的可溶性诱导表达条件，GST亲和层析与PSP柱上蛋白酶切纯化靶蛋白；
3.将纯化的YB-1免疫动物，制备其多克隆抗体；杂交瘤法制备YB-1单克隆抗体；进一步采用B细胞抗原表位预测法初步确定单抗识别的抗原表位；
4.用制备的YB-1多抗与单抗，建立血清YB-1定量化学发光免疫学检测方法（CLIA），并对建立的CLIA法进行方法学和检测性能评价；
5.以建立的CLIA定量测定健康人、良性与恶性肿瘤患者血清中YB-1蛋白水平，并初步评价其作为常见恶性肿瘤标志物的临床意义；
6.同时与甲胎蛋白的ROC曲线比较对原发性肝癌的诊断性能。
结果：
1.采用基因克隆技术成功构建了重组载体pGEX/YB-1；经表达、分离、纯化与蛋白酶切GST，获取重组YB-1蛋白；将其免疫动物制备了YB-1多抗；
2.建立了稳定分泌mAb且能特异识别YB-1的杂交瘤细胞株（1-D9、3-E8）；通过纯化制备了效价与亲和力较高的YB-1mAb；
3.杂交瘤细胞株1-D9和3-E8所分泌单抗的识别表位分别位于YB-1蛋白的（134-160aa）与（266-303aa）肽段；
4.建立了定量检测YB-1的CLIA检测方法；该法最低检测限为0.1μg/L，线性范围1.0-150.0μg/L，批内与批间变异系数分别为≤4.8和≤12.5%，平均回收率为100.6%，整个检测所需时间约90min；
5.初步确定了良性组（11.62±2.97μg/L）、恶性肿瘤组（18.69±6.11μg/L）以及健康组（10.79±2.39μg/L）血清YB-1水平；恶性肿瘤患者血清YB-1水平显著高于良性肿瘤患者及健康人（P<0.0001）；
6. ROC曲线显示，YB-1曲线下面积为0.896(P <0.0001)，在cut-off值≥13.45μg/L的条件下，血清YB-1辅助诊断恶性肿瘤的灵敏度为80%，特异性为81%；
7. ROC曲线分析表明，在诊断原发性肝癌上，YB-1的AUC为0.901（P <0.0001），在cut-off值＞14.69μg/L，血清YB-1辅助诊断原发性肝癌的灵敏度为84%，特异性为77%。而AFP的AUC为0.912（P <0.001），在cut-off值＞425.0μg/L，血清AFP辅助诊断原发性肝癌的灵敏度为78%，特异性为84%。
结论：
1.成功制备了重组YB-1蛋白及其多抗与单抗，并初步确定了单抗所识别的抗原表位；
2.成功建立了具较好检测性能的血清YB-1定量检测CLIA法；
3.血清YB-1水平可作为常见恶性肿瘤辅助诊断的标志物；
4.血清YB-1诊断原发性肝癌的效能与AFP接近；
5.该研究工作为进一步评价血清YB-1作为恶性肿瘤预后判断标志物的临床评价以及该法的临床推广应用提供了良好基础。
Objective:
1. Establishment of genetic engineering system to prepare recombinanthuman Y-box binding protein1(YB-1); and preparation of polyclonalantibody (pAb) and monoclonal antibody (mAb) against YB-1.
2. Development of a chemiluminescence immunoassay (CLIA) methodfor quantifying serum YB-1.
3. Quantitatifing serum YB-1with established CLIA, and investigatingthe clinical significance of serum YB-1as an aid-diagnostic biomarkerfor common malignancy tumors.
Methods:
1. YB-1coding sequence was obtained by RT-PCR using the total RNAof human renal cancer cell line786-0as template. The sequence wascloned into vector pGEX-6P-1and confirmed by double-enzymedigestion, and further identified by DNA sequencing.
2. For prokaryotic expression of YB-1, pGEX/YB-1was transformed into E. coli to express fusion protein GST-YB1. GST-affinitychromatography and chromatographic column protease digestion wereapplied to purify the recombinant YB-1protein.
3. PAb against human YB-1was raised by injecting a rabbitsubcutaneously four times at2-week intervals with2mg each time ofthe recombinant human YB-1. The IgG fraction of antiserum waspurified by protein A sepharose column chromatography. Anti-YB-1mAbs used in CLIA of human sera YB-1were prepared by hybridomatechnique; then B-lymphocytes epitope prediction were applied toidentify the epitopes recognition by YB-1mAbs.
4. The prepared pAb and mAb against YB-1were used to establish CLIAfor quantifying serum YB-1. Methodology evaluation was carried outto analysis the performance of CLIA.
5. The concentrations of serum YB-1in patients with malignant cancer(n=157), and benign tumor (n=30) and healthy individuals (n=100) ascontrol groups were determined by CLIA; and preliminary evaluationof clinical significance of serum YB-1as an aid-diagnostic tumormarker was performed.
6. Receiver operating characteristics (ROC) was used to compare thediagnostic performance of serum YB-1and alpha fetoprotein (AFP) fordiogmosis of hepatocellular carcinoma (HCC).
Results：
1. The vector pGEX/YB-1was successfully constructed and identified.The purified YB-1protein was prepared by means of prokaryoticexpression, GST-affinity chromatography and chromatographic columnprotease digestion.
2. YB-1pAb was obtained by immunizing rabbits.2hybridoma cell lines(1-D9,3-E8) stably secreting mAb against YB-1were obtained, thesemAbs could specifically recognize exogenous and endogenous YB-1.
3. The recognized epitope of the2mAbs might harbor in134-160aa and266-303aa of YB-1protein sequence.
4. CLIA was established based on double-antibody sandwich. Thecalibration curve of the CLIA had a coefficient of linear correlation r2>0.99(slope0.0702, intercept5.8112). The minimum detection limit was0.1μg/L; the linear range was from1.0to150.0μg/L; the intra-andinter-assay CV values were≤4.8and≤12.5%at10.0μg/L, respectively.The average recovery was100.6%(93.9%~109.0%). The total time ofthe assay was around90min.
5. With CLIA assay, the mean (SD) concentration of YB-1in100healthyserum samples was10.79±2.39μg/L; in30patients with benign tumorwas11.62±2.97μg/L; in157cancer patients was18.69±6.11μg/L,which was significantly higher than the values of healthy and benign tumor groups (P <0.0001).
6. ROC analysis demonstrated that the area under a ROC curve (AUC) forthe YB-1curve was0.896(P <0.0001). Serum YB-1had a sensitivityof80%(95%CI73%-86%), a specificity of81%(95%CI73%-87%)at the cut-off value of13.45μg/L for diagnosis cancer.
7. For diagnosis of HCC, the AUC for the YB-1curve was0.901(P <0.0001). Serum YB-1had a sensitivity of84%, a specificity of77%atthe cut-off value of14.69μg/L, while the AUC for the AFP curve was0.912(P <0.0001). AFP had a sensitivity of78%, a specificity of84%at the cut-off value of425.0μg/L.
Conclusions:
1. The recombinant YB-1protein and its pAbs and mAbs are successfullyprepared, and the recognized epitope of the two mAbs (1-D9,3-E8) isconfirmed.
2. A high-performance double antibody sandwich CLIA for thequantitative measurement of serum YB-1with the combination ofMAbs1-D9and Anti-YB-1PAbs has been developed.
3. Serum concentrations of YB-1might be used as a biomarker ofaided-diagnosis of common malignant tumors.
4. The diagnostic performance of serum YB-1is close to AFP for diagnosis of HCC.
5. Our primary exploratory study has suggested that quantifying serumYB-1, as an easily accessible circulating biomarker, could offer usefulaid-diagnostic information for patients with cancer; and this study laysfoundations for clinical application of the established CLIA in tumoraid-diagnosis and further evaluation of cancer prognosis.
引文
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[33] Oda Y, Ohishi Y, Basaki Y, et al. Prognostic implications of the nuclear localizationof Y-box-binding protein-1and CXCR4expression in ovarian cancer: theircorrelation with activated Akt, LRP/MVP and P-glycoprotein expression [J].Cancer Sci.2007;98:1020-1026.
[34] Kuwano M, Oda Y, Izumi H, et al. The role of nuclear Y-box binding protein1as aglobal marker in drug resistance [J]. Mol Cancer Ther.2004;3:1485-1492.
[35] Kohno K, Izumi H, Uchiumi T, Ashizuka M, Kuwano M. The pleiotropic functionsof the Y-box-binding protein, YB-1[J]. Bioessays.2003;25:691-698.
[36] Shibahara K, Sugio K, Osaki T, et al. Nuclear expression of the Y-box bindingprotein, YB-1, as a novel marker of disease progression in non-small cell lungcancer [J]. Clin Cancer Res.2001;7:3151-3155.
[37] Oda Y, Ohishi Y, Saito T, et al. Nuclear expression of Y-box-binding protein-1correlates with P-glycoprotein and topoisomerase II alpha expression, and withpoor prognosis in synovial sarcoma [J]. J Pathol.2003;199:251-258.
[1]李连弟，鲁凤珠，张思维等.中国恶性肿瘤死亡率20年变化趋势和近期预测分析[J].中华肿瘤杂志，1997，19：3~6.
[2] Scherf U, Ross DT, Waltham M, et al. A gene expression database for themolecular pharmacology of cancer [J]. Nat. Genet.2000,24:236~244.
[3]涂植光.依托现代科学管理和技术开展肿瘤标志物研究和应用[J]。中华检验医学杂志，2006，29：289~292.
[4] Kuwano M, Oda Y, Izumi H, et al. The role of nuclear Y-box binding protein1asa global marker in drug resistance [J]. Mol Cancer Ther,2004,3(11):1485~1492.
[5] Didier D K, Schiffenbauer J, Woulfe S L, Zacheis M, Schwartz B D.Characterization of the cDNA encoding a protein binding to the majorhistocompatibility complex class II Y box [J]. Proc Natl Acad Sci USA,1988,85(19):7322~7326.
[6] Karlson D, Nakaminami K, Toyomasu T. A Cold-regulated Nu-cleicAcid-binding Protien of Winter Wheat Shares a Domain with Bacterial ColdShock Proteins [J]. J Biol Chem,2002,277(38):35248~35256.
[7] Kohno K, Izumi H, Uchiumi T, Ashizuka M, Kuwano M. The pleiotropicfunctions of the Y-box-binding protein, YB-1[J]. Bioassays,2003,25:691~698.
[8] McCubrey JA, Steelman LS, Abrams SL, et al. Roles of the RAF/MEK/ERK andPI3K/PTEN/AKT pathways in malignant transformation and drug resistance [J].Adv Enzyme Regul.2006,46:249~279.
[9] Oda Y, Ohishi Y, Basaki Y, et al. Prognostic implications of the nuclearlocalization of Y-box-binding protein-1and CXCR4expression in ovarian cancer:their correlation with activated Akt, LRP/MVP and P-glycoprotein expression[J].Cancer Sci.2007,98(7):1020~1026.
[10] Bader AG and Vogt PK. Phosphorylation by Akt disables the anti-oncogenicactivity of YB-1[J]. Oncogene,2008,27:1179~1182.
[11] Basaki Y, Hosoi F, Oda Y, et al. Akt-dependent nuclear localization ofY-box-binding protein1in acquisition of malignant characteristics by humanovarian cancer cells [J]. Oncogene,2007,26(19):2736~2746.
[12] To K, Zhao Y, Jiang H, Hu K, et al. The phosphoinositide-dependent kinase-1inhibitor2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012) prevents Y-box binding protein-1frominducing epidermal growth factor receptor [J]. Mol Pharmacol.2007,72(3):641~652.
[13] Schittek B, Psenner K, Sauer B, et al. The increased expression of Y box-bindingprotein1in melanoma stimulates proliferation and tumor invasion, antagonizesapoptosis and enhances chemoresistance [J]. Int J Cancer.2007,120(10):2110~2118.
[14] Jurchott K, Bergmann S, Stein U, et al. YB-1as a cell cycle-regulatedtranscription factor facilitating cyclin A and cyclin B1gene expression [J]. JBiol Chem,2003,278(30):27988~27996.
[15] Gu C, Oyama T, Osaki T, et al. Expression of Y box-binding protein-1correlateswith DNA topoisomerase II alpha and proliferating cell nuclear antigenexpression in lung cancer [J]. Anticancer Res,2001,21(4A):2357~2362.
[16] Samuel S, Beifuss KK and Bernstein LR. YB-1binds to the MMP-13promotersequence and represses MMP-13transactivation via the AP-1site. Biochimicaet biophysica Acta (BBA)–Gene Structure and Expression [J].2007,1769:525~531.
[17] Lasham A, Lindridge E, Rudert F, et al. Regulation of the human fas promoterby YB-1, Puralpha and AP-1transcription factors [J]. Gene,2000,252(1~2):1~13.
[18] Maruyama Y, Ono M, Kawahara A, et al. Tumor growth suppression inpancreatic cancer by a putative metastasis suppressor gene Cap43/NDRG1/Drg-1through modulation of angiogenesis [J]. Cancer Res.2006,66(12):6233~6242.
[19] Fujii T, Yokoyama G, Takahashi H, Namoto R, Nakagawa S, Toh U, Kage M,Shirouzu K, Kuwano M. Preclinical studies of molecular-targeting diagnostic andtherapeutic strategies against breast cancer [J]. Breast Cancer.2008,15(1):73~78.
[20] Guay D, Gaudreault I, Massip L, et al. Formation of a nuclear complexcontaining the p53tumor suppressor, YB-1, and the Werner syndrome geneproduct in cells treated with UV light [J]. Int J Biochem Cell Biol,2006,38(8):1300~1313.
[21] Stratford AL, Habibi G, Astanehe A, et al. Epidermal growth factor receptor(EGFR) is transcriptionally induced by the Y-box binding protein-1(YB-1) andcan be inhibited with Iressa in basal-like breast cancer, providing a potentialtarget for therapy [J]. Breast Cancer Res.2007,9(5):R61.
[22] Lutz M, Wempe F, Bahr I, et al. Proteasomal degradation of the multifunctionalregulator YB-1is mediated by an F-Box protein induced during programmed celldeath [J]. FEBS Lett,2006,580(16):3921~3930.
[23] Sutherland BW, Kucab J, Wu J, et al. Akt phosphorylates the Y-box bindingprotein1at Ser102located in the cold shock domain and affects theanchorage-independent growth of breast cancer cells [J]. Oncogene,2005,24(26):4281~4292.
[24] Loo TW, Clarke DM. Recent progress in understanding the mechanism ofP-glycoprotein-mediated drug efflux [J]. J Membr Biol,2005,206:173~185.
[25] Sisodiya SM, Martinian L, Scheffer GL, et al. Vascular colocalization ofP-glycoprotein, multidrug-resistance associated protein1, breast cancerresistance protein and major vault protein in human epileptogenic pathologies [J].Neuropathol Appl Neurobiol,2006,32:51~63
[26] Callaghan R, Ford RC, Kerr ID. The translocation mechanism of P-glycoprotein[J]. FEBS Lett,2006,580:1056~1063.
[27] Torigoe T, Izumi H, Ishiguchi H, et al. Cisplatin Resistance and TranscriptionFactors [J]. Curr Med Chem-Anti-Cancer Agents,2005,5,15~27.
[28] Shibahara K, Uchiumi T, et al. Targeted disruption of one allele of the Y-boxbinding protein-1(YB-1) gene in mouse embryonic stem cells and increasedsensitivity to cisplatin and mitomycin C [J]. Cancer Sci,2004,95(4):348~353.
[29] Mantwill K, K hler-Vargas N, Bernshausen A, et al. Inhibition of themultidrug-resistant phenotype by targeting YB-1with a conditionally oncolyticadenovirus: implications for combinatorial treatment regimen withchemotherapeutic agents [J]. Cancer Res.2006,66(14):7195~202.
[30] Bargou RC, Jurchott K, Wagener C, et al. Nuclear localization and increasedlevels of transcription factor YB-1in primary human breast cancers areassociated with intrinsic MDR1gene expression [J]. Nat Med,1997,3(4):447~450.
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[32] Stein U, Bergmann S, Scheffer G L, et al. YB-1facilitates basal and5-fluorouracil-inducible expression of the human major vault protein (MVP)gene[J]. Oncogene,2005,24(22):3606~3618.
[33] Shibao K, Takano H, Nakayama Y, et al. Enhanced coexpression of YB-1andDNA topoisomerase II alpha genes in human colorectal carcinomas[J]. Int JCancer,1999,83(6):732~737.
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[1]李连弟，鲁凤珠，张思维等.中国恶性肿瘤死亡率20年变化趋势和近期预测分析[J].中华肿瘤杂志，1997，19：3~6.
[2] Scherf U, Ross DT, Waltham M, et al. A gene expression database for themolecular pharmacology of cancer [J]. Nat. Genet.2000,24:236~244.
[3]涂植光.依托现代科学管理和技术开展肿瘤标志物研究和应用[J]。中华检验医学杂志，2006，29：289~292.
[4] Kuwano M, Oda Y, Izumi H, et al. The role of nuclear Y-box binding protein1asa global marker in drug resistance [J]. Mol Cancer Ther,2004,3(11):1485~1492.
[5] Didier D K, Schiffenbauer J, Woulfe S L, Zacheis M, Schwartz B D.Characterization of the cDNA encoding a protein binding to the majorhistocompatibility complex class II Y box [J]. Proc Natl Acad Sci USA,1988,85(19):7322~7326.
[6] Karlson D, Nakaminami K, Toyomasu T. A Cold-regulated Nu-cleicAcid-binding Protien of Winter Wheat Shares a Domain with Bacterial ColdShock Proteins [J]. J Biol Chem,2002,277(38):35248~35256.
[7] Kohno K, Izumi H, Uchiumi T, Ashizuka M, Kuwano M. The pleiotropicfunctions of the Y-box-binding protein, YB-1[J]. Bioassays,2003,25:691~698.
[8] McCubrey JA, Steelman LS, Abrams SL, et al. Roles of the RAF/MEK/ERK andPI3K/PTEN/AKT pathways in malignant transformation and drug resistance [J].Adv Enzyme Regul.2006,46:249~279.
[9] Oda Y, Ohishi Y, Basaki Y, et al. Prognostic implications of the nuclearlocalization of Y-box-binding protein-1and CXCR4expression in ovarian cancer:their correlation with activated Akt, LRP/MVP and P-glycoprotein expression[J].Cancer Sci.2007,98(7):1020~1026.
[10] Bader AG and Vogt PK. Phosphorylation by Akt disables the anti-oncogenicactivity of YB-1[J]. Oncogene,2008,27:1179~1182.
[11] Basaki Y, Hosoi F, Oda Y, et al. Akt-dependent nuclear localization ofY-box-binding protein1in acquisition of malignant characteristics by humanovarian cancer cells [J]. Oncogene,2007,26(19):2736~2746.
[12] To K, Zhao Y, Jiang H, Hu K, et al. The phosphoinositide-dependent kinase-1inhibitor2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012) prevents Y-box binding protein-1frominducing epidermal growth factor receptor [J]. Mol Pharmacol.2007,72(3):641~652.
[13] Schittek B, Psenner K, Sauer B, et al. The increased expression of Y box-bindingprotein1in melanoma stimulates proliferation and tumor invasion, antagonizesapoptosis and enhances chemoresistance [J]. Int J Cancer.2007,120(10):2110~2118.
[14] Jurchott K, Bergmann S, Stein U, et al. YB-1as a cell cycle-regulatedtranscription factor facilitating cyclin A and cyclin B1gene expression [J]. JBiol Chem,2003,278(30):27988~27996.
[15] Gu C, Oyama T, Osaki T, et al. Expression of Y box-binding protein-1correlateswith DNA topoisomerase II alpha and proliferating cell nuclear antigenexpression in lung cancer [J]. Anticancer Res,2001,21(4A):2357~2362.
[16] Samuel S, Beifuss KK and Bernstein LR. YB-1binds to the MMP-13promotersequence and represses MMP-13transactivation via the AP-1site. Biochimicaet biophysica Acta (BBA)–Gene Structure and Expression [J].2007,1769:525~531.
[17] Lasham A, Lindridge E, Rudert F, et al. Regulation of the human fas promoterby YB-1, Puralpha and AP-1transcription factors [J]. Gene,2000,252(1~2):1~13.
[18] Maruyama Y, Ono M, Kawahara A, et al. Tumor growth suppression inpancreatic cancer by a putative metastasis suppressor gene Cap43/NDRG1/Drg-1through modulation of angiogenesis [J]. Cancer Res.2006,66(12):6233~6242.
[19] Fujii T, Yokoyama G, Takahashi H, Namoto R, Nakagawa S, Toh U, Kage M,Shirouzu K, Kuwano M. Preclinical studies of molecular-targeting diagnostic andtherapeutic strategies against breast cancer [J]. Breast Cancer.2008,15(1):73~78.
[20] Guay D, Gaudreault I, Massip L, et al. Formation of a nuclear complexcontaining the p53tumor suppressor, YB-1, and the Werner syndrome geneproduct in cells treated with UV light [J]. Int J Biochem Cell Biol,2006,38(8):1300~1313.
[21] Stratford AL, Habibi G, Astanehe A, et al. Epidermal growth factor receptor(EGFR) is transcriptionally induced by the Y-box binding protein-1(YB-1) andcan be inhibited with Iressa in basal-like breast cancer, providing a potentialtarget for therapy [J]. Breast Cancer Res.2007,9(5):R61.
[22] Lutz M, Wempe F, Bahr I, et al. Proteasomal degradation of the multifunctionalregulator YB-1is mediated by an F-Box protein induced during programmed celldeath [J]. FEBS Lett,2006,580(16):3921~3930.
[23] Sutherland BW, Kucab J, Wu J, et al. Akt phosphorylates the Y-box bindingprotein1at Ser102located in the cold shock domain and affects theanchorage-independent growth of breast cancer cells [J]. Oncogene,2005,24(26):4281~4292.
[24] Loo TW, Clarke DM. Recent progress in understanding the mechanism ofP-glycoprotein-mediated drug efflux [J]. J Membr Biol,2005,206:173~185.
[25] Sisodiya SM, Martinian L, Scheffer GL, et al. Vascular colocalization ofP-glycoprotein, multidrug-resistance associated protein1, breast cancerresistance protein and major vault protein in human epileptogenic pathologies [J].Neuropathol Appl Neurobiol,2006,32:51~63
[26] Callaghan R, Ford RC, Kerr ID. The translocation mechanism of P-glycoprotein[J]. FEBS Lett,2006,580:1056~1063.
[27] Torigoe T, Izumi H, Ishiguchi H, et al. Cisplatin Resistance and TranscriptionFactors [J]. Curr Med Chem-Anti-Cancer Agents,2005,5,15~27.
[28] Shibahara K, Uchiumi T, et al. Targeted disruption of one allele of the Y-boxbinding protein-1(YB-1) gene in mouse embryonic stem cells and increasedsensitivity to cisplatin and mitomycin C [J]. Cancer Sci,2004,95(4):348~353.
[29] Mantwill K, K hler-Vargas N, Bernshausen A, et al. Inhibition of themultidrug-resistant phenotype by targeting YB-1with a conditionally oncolyticadenovirus: implications for combinatorial treatment regimen withchemotherapeutic agents [J]. Cancer Res.2006,66(14):7195~202.
[30] Bargou RC, Jurchott K, Wagener C, et al. Nuclear localization and increasedlevels of transcription factor YB-1in primary human breast cancers areassociated with intrinsic MDR1gene expression [J]. Nat Med,1997,3(4):447~450.
[31] Ohga T, Koike K, Ono M, et al. Role of the human Y box-binding protein YB-1in cellular sensitivity to the DNA-damaging agents cisplatin, mitomycin C, andultraviolet light [J]. Cancer Res,1996,56(18):4224~4228.
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