替莫唑胺耐药的恶性胶质瘤细胞系耐药特性演变的研究
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摘要
胶质母细胞瘤(WHOⅣ)是颅内最常见的高恶性肿瘤,具有弥漫浸润性生长的生物学特性,预后不佳。尽管以手术为主联合放射及化学治疗的综合方案已成为治疗指南,但胶质母细胞瘤病人的预后并没有得到明显改善,平均生存时间只有12～15个月。肿瘤细胞对化疗药物的耐药性是导致治疗失败的主要原因之一,耐药涉及因素很多,其中O6-甲基鸟嘌呤-DNA甲基转移酶(O6-methylguanine-DNA methyltransferase, MGMT)与烷化剂耐药之间的关系最受关注。MGMT是一种DNA修复酶,其可在不需任何辅助因子或其他蛋白质的条件下催化DNA分子中鸟嘌呤O6位上的烷基转移至MGMT本身第145位的半胱氨酸残基上,鸟嘌呤得以复原,DNA的结构和功能得以恢复从而保护细胞免受烷化剂的损伤。
高脂溶性和易于透过血脑屏障的烷化剂,被国内外公认为治疗胶质瘤的标准化疗药物。替莫唑胺(Temozolomide, TMZ)属于第二代烷化剂化疗药,是咪唑四嗪类衍生物,不经肝脏代谢,直接在脑胶质瘤组织碱性环境中分解,生成活性物质MTIC (3-methyl-triazenl-yl) imidazole-4-carboxomide, MTIC),疗效佳,副作用小,目前备受临床青睐。然而,肿瘤细胞固有的或获得性的耐药部分地限制了替莫唑胺的临床应用效果。
沙利度胺(Thalidomide,THD),是谷氨酸类衍生物,具有抗血管生成作用,鉴于胶质母细胞瘤富含血管,因此沙利度胺被认为具有治疗恶性胶质瘤的潜力。在多项Ⅱ期临床试验中,替莫唑胺联合沙利度胺的化疗方案可以明显延长恶性胶质瘤病人的中位生存期,无明显的毒副作用。
对沙利度胺联合替莫唑胺治疗胶质瘤的具体机制逐渐明确,在我们前期实验中发现沙利度胺可以通过激活胶质瘤细胞中PTEN的表达,抑制PI3K/AKT/mTOR信号通路的激活,促进替莫唑胺诱导的自噬及自噬性死亡,且两者联合可以通过激活caspase-3,诱导凋亡发生。然而,目前尚无替莫唑胺联合沙利度胺对替莫唑胺耐药的胶质母细胞瘤是否有作用的研究报道。
本课题研究分为以下二个部分：
第一部分探讨胶质瘤细胞耐药性演变规律及TMZ自身改变耐药细胞耐药特性的机理,以期为临床优化化疗方案提供依据。我们通过体外分步诱导法使人脑胶质瘤U251细胞对TMZ (0.25-16μg/mL)产生耐药；用MTT法检测耐药指数及细胞存活率；用Western blot、RT-PCR、免疫组化及免疫荧光方法检测耐药机理及增加TMZ浓度后导致胶质瘤耐药细胞生长受抑制的可能原因。历经8个月成功建立了对TMZ耐药的U251/TR, U251/TR细胞IC50 (220.87μM)是未经诱导U251细胞IC50 (33.12μM)的6.67倍(P<0.01),其耐药指数(RF)约为7；U251／TR细胞MGMT表达较未经诱导U251细胞明显增加(P<0.01)；同时本实验发现倍增替莫唑胺浓度仍可以使耐药细胞生长出现受抑制现象,U251／TR细胞MGMT检测结果显示其表达有相应下降。
第二部分探讨沙利度胺联合替莫唑胺是否对U251／TR耐药细胞耐药特性有影响,并研究其可能机制。用MTT法检测耐药指数及细胞存活率；用Western blot、RT-PCR、免疫荧光等方法检测蛋白表达水平；用甲基化特异性PCR检测MGMT启动子甲基化情况。给药24小时后,双药联合组的细胞存活率明显降低,48小时后替莫唑胺组细胞存活率也开始下降,且前者降低更明显,沙利度胺组基本无下降；双药联合组及替莫唑胺组MGMT蛋白下降,两组下降水平无差异；甲基化特异性PCR结果显示启动子区甲基化情况无改变。
结论：
1. MGMT表达水平增高是导致U251／TR对TMZ耐药的主要机制；替莫唑胺可以通过消耗MGMT,从而改变耐药细胞耐药特性,起到自身克服耐药作用。这为临床优化节律性化疗方案提供了依据。
2.沙利度胺联合替莫唑胺可以更好的抑制耐药细胞的增殖,起到较好的化疗作用,其机制不是通过改变MGMT基因启动子区甲基化状态来实现,具体机制尚需进一步研究证实。
Glioblastoma (GBM WHOⅣ) is the most common and malignant intracranial tumor with diffusely infiltrative growth characteristics and dismal prognosis. The median survival of patients ranges from 12 to 15 months despite of aggressive surgery combined with radiation and chemotherapy. The highly lethal nature of this tumor partly accounts for its chemoresistance of GBM cells. The resistance of GBM cells to alkylating agents is mediated by many factors. Among these factors, the association between O6-methylguanine-DNA methyltransferase (MGMT) and chemo-resistance is more concerned. MGMT is a key enzyme in the DNA repair network, which catalyzes the transfer of the methyl group from O6-methylguanine to a cysteine residue of its active site. In this single step reaction, DNA-lesions caused by alkylating substances are repaired. MGMT subsequently is irreversibly inactivated and degraded.
Although advances in chemotherapeutic drugs, alkylating agents remain the most common and effective administration in GBM, based on their lipophilic nature, which can appreciably permeate blood-brain barrier (BBB), and their intrinsic cytotoxic activities. Temozolomide (TMZ), an orally administered cytotoxic alkylating agent with broad-spectrum antitumor activity, is a bioavailable imidazotetrazine derivative of the alkylating agent dacarbazine. It penetrates BBB into the brain easily and undergoes rapid chemical conversion in the systemic circulation at physiological pH to its active compound, MTIC (5-(3-methyltriazen-l-yl)imidazole-4-carboximide), which degrades to a DNA-alkylating species, subsequently, is cytotoxic via the formation of O6-and N7-alkylguanine lesions in DNA, this procedure does not require hepatic metabolism for activation. TMZ becomes the most perspective chemotherapeutic drug against gliomas, on base that it is more effective, easier to administer and has fewer side-effects than its competitors. However, it has also failed to improve prognosis in all patients with GBM. The unsatisfactory outcome with chemotherapy has chiefly been originated from GBM tumor cells intrinsic or acquired chemoresistance.
Thalidomide, a glutamic acid derivative, is considered as a potential drug to treat hyper-vascularized glioma due to its anti-angiogenesis effect. In a number of phase II clinical trials, thalidomide combined with temozolomide has been proved to prolong the median survival of patients with malignant glioma significantly, meanwhile, the side effect of this combination is not obvious.
The mechanism of combination of thalidomide combined with temozolomide to treat gliomas is cleared gradually. In our previous study, we found thalidomide may inhibit PI3K/AKT/mTOR signaling pathway through activation of PTEN, a tumor suppress gene, to reinfore the induced autophagy. Meanwhile, the combination of both may induce apoptosis through activation of caspase-3. Furthermore, there is no study to examine whether the resistant characteristic of the TMZ-resistant human glioma cell line could be changed by temozolomide combined with thalidomide.
The present study was divided into two parts.
In the first part of this study was aimed to investigate how and why the chemoresistance to TMZ evolved in U251/TR cells, a TMZ-resistant cell line under TMZ administrator, and provide experimental evidence for optimal TMZ therapy strategy. U251/TR, was established by stepwise exposure of human U251 parental cells to TMZ induced culture for 8 months. MTT assay was used to calculate the resistance index and cell viability percentage. Western blotting(WB)、RT-PCR、immunohistochemistry (IHC) and immunofluorescence(IF) were used to detect MGMT expression for the analysis of chemoresistant evolution and its mechanism in U251 cells during the TMZ induction procedure. U251/TR showed approximately 7-fold resistance to TMZ (RF=6.67±0.53). The MGMT protein expression was significantly increased in U251/TR cells compared with the parental U251 cells (p<0.01). The growth of U251/TR cells was inhibited significantly by TMZ with double the final induced strength from day 3 to 5 compared with DMSO control group (p<0.01, for all) and MGMT protein level in those treated cells was decreased accordingly after TMZ administration (p<0.05).
The second part focused on whether the resistant characteristic of the TMZ-resistant human glioma cell line could be changed by thalidomide combined with temozolomide. MTT assay was used to calculate the resistance index and cell viability percentage. Western blotting(WB)、RT-PCR、and immunofluorescence (IF) were used to detect MGMT expression for the analysis of U251/TR cells treated with temozolomide combined with thalidomide. MGMT promoter profile was analyzed by Methylation-specific polymerase-Chain-reaction analysis (MSP). Compared to negative control group, significant inhibition of proliferation was shown in TMZ+THD group after 24 h and TMZ group after 48 h. The MGMT protein levels in TMZ+THD group and TMZ group decreased significantly (p<0.01), moreover, there is no difference between both groups. We also found no change in promoter methylation in all groups.
Conclusions
1. Our results demonstrated that the primary mechanism of U251/TR cells resistant to TMZ is owing to increased activity of MGMT and the chemoresistance was evolving during the TMZ induction procedure. TMZ could overcome the TMZ-resistance itself by consuming MGMT steadily, which suggests that metronomic TMZ regimen could improve the chemo-sensitivity in TMZ resistant glioma clinically.
2. Thalidomide combined with temozolomide could improve the cytotoxicity in U251/TR cells. The mechanism needs further evaluation in future prospective trials.
高脂溶性和易于透过血脑屏障的烷化剂,被国内外公认为治疗胶质瘤的标准化疗药物。替莫唑胺(Temozolomide, TMZ)属于第二代烷化剂化疗药,是咪唑四嗪类衍生物,不经肝脏代谢,直接在脑胶质瘤组织碱性环境中分解,生成活性物质MTIC (3-methyl-triazenl-yl) imidazole-4-carboxomide, MTIC),疗效佳,副作用小,目前备受临床青睐。然而,肿瘤细胞固有的或获得性的耐药部分地限制了替莫唑胺的临床应用效果。
沙利度胺(Thalidomide,THD),是谷氨酸类衍生物,具有抗血管生成作用,鉴于胶质母细胞瘤富含血管,因此沙利度胺被认为具有治疗恶性胶质瘤的潜力。在多项Ⅱ期临床试验中,替莫唑胺联合沙利度胺的化疗方案可以明显延长恶性胶质瘤病人的中位生存期,无明显的毒副作用。
对沙利度胺联合替莫唑胺治疗胶质瘤的具体机制逐渐明确,在我们前期实验中发现沙利度胺可以通过激活胶质瘤细胞中PTEN的表达,抑制PI3K/AKT/mTOR信号通路的激活,促进替莫唑胺诱导的自噬及自噬性死亡,且两者联合可以通过激活caspase-3,诱导凋亡发生。然而,目前尚无替莫唑胺联合沙利度胺对替莫唑胺耐药的胶质母细胞瘤是否有作用的研究报道。
本课题研究分为以下二个部分：
第一部分探讨胶质瘤细胞耐药性演变规律及TMZ自身改变耐药细胞耐药特性的机理,以期为临床优化化疗方案提供依据。我们通过体外分步诱导法使人脑胶质瘤U251细胞对TMZ (0.25-16μg/mL)产生耐药；用MTT法检测耐药指数及细胞存活率；用Western blot、RT-PCR、免疫组化及免疫荧光方法检测耐药机理及增加TMZ浓度后导致胶质瘤耐药细胞生长受抑制的可能原因。历经8个月成功建立了对TMZ耐药的U251/TR, U251/TR细胞IC50 (220.87μM)是未经诱导U251细胞IC50 (33.12μM)的6.67倍(P<0.01),其耐药指数(RF)约为7；U251／TR细胞MGMT表达较未经诱导U251细胞明显增加(P<0.01)；同时本实验发现倍增替莫唑胺浓度仍可以使耐药细胞生长出现受抑制现象,U251／TR细胞MGMT检测结果显示其表达有相应下降。
第二部分探讨沙利度胺联合替莫唑胺是否对U251／TR耐药细胞耐药特性有影响,并研究其可能机制。用MTT法检测耐药指数及细胞存活率；用Western blot、RT-PCR、免疫荧光等方法检测蛋白表达水平；用甲基化特异性PCR检测MGMT启动子甲基化情况。给药24小时后,双药联合组的细胞存活率明显降低,48小时后替莫唑胺组细胞存活率也开始下降,且前者降低更明显,沙利度胺组基本无下降；双药联合组及替莫唑胺组MGMT蛋白下降,两组下降水平无差异；甲基化特异性PCR结果显示启动子区甲基化情况无改变。
结论：
1. MGMT表达水平增高是导致U251／TR对TMZ耐药的主要机制；替莫唑胺可以通过消耗MGMT,从而改变耐药细胞耐药特性,起到自身克服耐药作用。这为临床优化节律性化疗方案提供了依据。
2.沙利度胺联合替莫唑胺可以更好的抑制耐药细胞的增殖,起到较好的化疗作用,其机制不是通过改变MGMT基因启动子区甲基化状态来实现,具体机制尚需进一步研究证实。
Glioblastoma (GBM WHOⅣ) is the most common and malignant intracranial tumor with diffusely infiltrative growth characteristics and dismal prognosis. The median survival of patients ranges from 12 to 15 months despite of aggressive surgery combined with radiation and chemotherapy. The highly lethal nature of this tumor partly accounts for its chemoresistance of GBM cells. The resistance of GBM cells to alkylating agents is mediated by many factors. Among these factors, the association between O6-methylguanine-DNA methyltransferase (MGMT) and chemo-resistance is more concerned. MGMT is a key enzyme in the DNA repair network, which catalyzes the transfer of the methyl group from O6-methylguanine to a cysteine residue of its active site. In this single step reaction, DNA-lesions caused by alkylating substances are repaired. MGMT subsequently is irreversibly inactivated and degraded.
Although advances in chemotherapeutic drugs, alkylating agents remain the most common and effective administration in GBM, based on their lipophilic nature, which can appreciably permeate blood-brain barrier (BBB), and their intrinsic cytotoxic activities. Temozolomide (TMZ), an orally administered cytotoxic alkylating agent with broad-spectrum antitumor activity, is a bioavailable imidazotetrazine derivative of the alkylating agent dacarbazine. It penetrates BBB into the brain easily and undergoes rapid chemical conversion in the systemic circulation at physiological pH to its active compound, MTIC (5-(3-methyltriazen-l-yl)imidazole-4-carboximide), which degrades to a DNA-alkylating species, subsequently, is cytotoxic via the formation of O6-and N7-alkylguanine lesions in DNA, this procedure does not require hepatic metabolism for activation. TMZ becomes the most perspective chemotherapeutic drug against gliomas, on base that it is more effective, easier to administer and has fewer side-effects than its competitors. However, it has also failed to improve prognosis in all patients with GBM. The unsatisfactory outcome with chemotherapy has chiefly been originated from GBM tumor cells intrinsic or acquired chemoresistance.
Thalidomide, a glutamic acid derivative, is considered as a potential drug to treat hyper-vascularized glioma due to its anti-angiogenesis effect. In a number of phase II clinical trials, thalidomide combined with temozolomide has been proved to prolong the median survival of patients with malignant glioma significantly, meanwhile, the side effect of this combination is not obvious.
The mechanism of combination of thalidomide combined with temozolomide to treat gliomas is cleared gradually. In our previous study, we found thalidomide may inhibit PI3K/AKT/mTOR signaling pathway through activation of PTEN, a tumor suppress gene, to reinfore the induced autophagy. Meanwhile, the combination of both may induce apoptosis through activation of caspase-3. Furthermore, there is no study to examine whether the resistant characteristic of the TMZ-resistant human glioma cell line could be changed by temozolomide combined with thalidomide.
The present study was divided into two parts.
In the first part of this study was aimed to investigate how and why the chemoresistance to TMZ evolved in U251/TR cells, a TMZ-resistant cell line under TMZ administrator, and provide experimental evidence for optimal TMZ therapy strategy. U251/TR, was established by stepwise exposure of human U251 parental cells to TMZ induced culture for 8 months. MTT assay was used to calculate the resistance index and cell viability percentage. Western blotting(WB)、RT-PCR、immunohistochemistry (IHC) and immunofluorescence(IF) were used to detect MGMT expression for the analysis of chemoresistant evolution and its mechanism in U251 cells during the TMZ induction procedure. U251/TR showed approximately 7-fold resistance to TMZ (RF=6.67±0.53). The MGMT protein expression was significantly increased in U251/TR cells compared with the parental U251 cells (p<0.01). The growth of U251/TR cells was inhibited significantly by TMZ with double the final induced strength from day 3 to 5 compared with DMSO control group (p<0.01, for all) and MGMT protein level in those treated cells was decreased accordingly after TMZ administration (p<0.05).
The second part focused on whether the resistant characteristic of the TMZ-resistant human glioma cell line could be changed by thalidomide combined with temozolomide. MTT assay was used to calculate the resistance index and cell viability percentage. Western blotting(WB)、RT-PCR、and immunofluorescence (IF) were used to detect MGMT expression for the analysis of U251/TR cells treated with temozolomide combined with thalidomide. MGMT promoter profile was analyzed by Methylation-specific polymerase-Chain-reaction analysis (MSP). Compared to negative control group, significant inhibition of proliferation was shown in TMZ+THD group after 24 h and TMZ group after 48 h. The MGMT protein levels in TMZ+THD group and TMZ group decreased significantly (p<0.01), moreover, there is no difference between both groups. We also found no change in promoter methylation in all groups.
Conclusions
1. Our results demonstrated that the primary mechanism of U251/TR cells resistant to TMZ is owing to increased activity of MGMT and the chemoresistance was evolving during the TMZ induction procedure. TMZ could overcome the TMZ-resistance itself by consuming MGMT steadily, which suggests that metronomic TMZ regimen could improve the chemo-sensitivity in TMZ resistant glioma clinically.
2. Thalidomide combined with temozolomide could improve the cytotoxicity in U251/TR cells. The mechanism needs further evaluation in future prospective trials.
引文
[1]. Palanichanmy K, Erkkinen M, Chakravarti A. Predictive and prognostic markers in human glioblastomas. J Curr Teat Options Oncol,2006,7:490-504.
[2]潘强,杨学军,高松等.替莫唑胺改变人U251胶质瘤细胞系耐药特性机制的体外研究[J].中国现代神经疾病杂志,2009,9(6)：583-588.
[3]GAO Song, YANG Xue-jun, ZHANG Wen-gao, et al. Mechanism of thalidomide to enhance cytotoxicity of temozolomide in U251-MG glioma cells in vitro. Chin Med J,2009; 122 (11):1260-1266.
[4]Nicholas GA, Tracy TB. New Treatment Strategies for Malignant Gliomas. Oncologist.1999;4:209-224
[5]Mineura K, Yanagisawa T, Watanabe K, et al. Human brain tumor O6-methylaguanine-DNA methyltransferase mRNA and its significance as indicator of selective chloroethylnitrosourea chemotherapy. Int J Cancer.1996, 69:420-425.
[6]Paus C, Murat A, Stupp R,et al. Role of MGMT and clinical applications in brain tumors. J Bull Cancer.2007,94(9):769-73.
[7]Bearzaatto A,S zadkowski M, Macpherson P, et al. Epigenetic regulation of the MGMT and hMSH6 DNA repair genes in cells resistant to methylating agents. Cancer Res,2000,60 (12):3262-3270.
[8]Teo SK, Stirling DI, Zeldis JB. Thalidomide as a novel therapeutic agent:new uses for an old produt. Drug Discov Today.2005,10:104-107.
[9]Chang SM, Lamborn KR, Malec M, et al. Phase Ⅱ study of temozolomide and thalidomide with radiation therapy for newly diagnosed glioblastoma multiforme. Int J Radiat Oncol Biol Phys.2004,60(2):353-357.
[10]Puduvalli VK, Yung WK, Hess KR, et al.Phase Ⅱ study of fenretinide (NSC 374551) in adults with recurrent malignant gliomas:A North American Brain Tumor Consortium study. J Clin Oncol.2004,22(21):4282-4289.
[11]Baumann F, Bjeljac M, Kollias SS, et al. Combined thalidomide and temozolomide treatment in patients with glioblastoma multiforme. J Neurooncol.2004, 67(1-2):191-200.
[12]常洪波,章翔,甄海宁,等.白藜芦醇抑制SHG-44胶质瘤细胞生长实验研究 [J].中华神经外科疾病研究杂志,2006,5(2)：119-123.
[13]李晓兴,王之敏,左剑玲等.替莫唑胺诱导的人脑胶质瘤耐药细胞系的建立及其耐药性逆转.苏州大学学报(医学版)2007,27：169-171.
[14]Kanzawa T, Germano IM,Komata T,et al. Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ. 2004,11:448-457.
[15]Kim JD, Choi BK, Bae JS, et al. Clonin and characterization of GITR ligand [J]. Genes Immun,2003,4(8):564-569.
[16]王伯沄.免疫荧光组织化学和荧光组织化学技术。第四军医大学科技资料增刊,1974,2(1)：5.
[17]周庚寅主编.组织病理学技术[M].北京：北京大学医学出版社,2006：58.
[18]Markus B.Anticancer drug resistence in primary human brain tumors. Brain Res Rev.2001,35:161-204.
[19]Uesaka T, Shono T, Kuga D,et al. Enhanced expression of DNA topoisomerase Ⅱ genes in human medulloblastoma and its possible association with etoposide sensitivity. J Neuro-oncol.2007,84(2):119-29. Epub 2007 Mar 15.
[20]Monzo M, Rosell R, Taron M. Drug resistance in non-small cell lung cancer [J]. Lung cancer,2001,34(Suppl 2):S91-94.
[21]Spiegl-Kreinecker S, Buchroithner J, Elbling L, et al. Expression and functional activity of the ABC-transporter proteins P-glycoprotein and multidrug-resistance protein 1 in human brain tumor cells and astrocytes. J Neurooncol.2002, 57(1):27-36.
[22]Bahr O, Rieger J, Duffner F, et al. P-glycoprotein and multidrug resistance-associated protein mediate specific patterns of multidrug resistance in malignant glioma cell lines, but not in primary glioma cells. Brain Pathol. 2003,13(4):482-94.
[23]Cole SP, Deeley RG. Multidrug resistance mediated by the ATP-binding cassette trans-porter protein MRP. Bioessays.1998,20(11):931-40.
[24]Benyahia B, Huguet S, Decleves X, et al. Multidrug resistance-associated protein MRP1 expression in human gliomas:chemosensitization to vincristine and etoposide by indome-thacin in human glioma cell lines overexpressing MRP1. J Neurooncol.2004,66 (1-2):65-70.
[25]Sisodiya SM, Martinian L, Scheffer GL, et al. Major vault protein, a marker of drug resistance, is upregulated in refractory epilepsy. Epilepsia.2003, 44(11):1388-96.
[26]Sasaki T, Hankins GR, Helm GA. Major vault protein/lung resistance-related protein (MVP/LRP) expression in nervoussystem tumors. Brain Tumor Path 2002, 19:59-62.
[27]Pollack I F, Kwaecki S, Lazo J S. Blocking of glioma proliferation in vitro and in vivo and potentiating the effects of BCNU and cisplatin:UCN-01, a selective protein kinase Cinhibitor, J Neurosurg.1996,84(6):1024-1032.
[28]Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci USA.1998,95:15665-70.
[29]Zhang W, Mojsilovic-Petrovic J, Andrade MF, et al. The expression and functional characterization of ABCG2 in brain endothelial cells and vessels. FASEB J.2003, 17(14):2085-7. Epub 2003 Sep 4.
[30]Iwadate Y, Fujimoto S, Tagawa M, et al. Association of p53 gene muation with decreased chemosensitivity in human malignant gliomas. Int J Cancer.1996, 69(3):236-40.
[31]Fojo T, Bates S. Strategies for reversing drug resistance. Oncogene,2003, 22(47):7512.
[32]Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer,2006,5:67-71.
[33]Shervington A, Lu C. Expression of multidrug resistance genes in normal and cancer stem cells. Czncer Invest,2008,26:55-542.
[34]Bi CL, Fang JS, Chen FH, et al. Chemoresistance of CD133(+) tumor stem cells from human brain glioma. Zhong Nan Da Xue Xue Bao Yi Xue Ban.2007, 32(4):568-73.
[35]Caircross JG, Ueki K, Zlatescu MC, et al. Specific genetic predictors of chemotherapeutic response and survival in patients with anaplastic oligodendrogliomas. J Natl Cancer Res,2003,9(4):1461-1468.
[36]Ito M, Wakabayashi T, Natsume A, et al. Genetically heterogeneous glioblastoma recurring with disappearance of lp/19q losses:case report. Neurosurgery.2007, 61(1):E168-9; discussion E169.
[37]潘强,杨学军.胶质瘤化疗耐药分子机制研究进展.中国临床神经外科杂志,2010,15(2)：118-122.
[38]RI弗雷谢尼,动物细胞培养基本技术指南(分子克隆实验指南系列).第4版.北京：科学出版社,2004：398-400.
[39]Friedman HS, Kokkinakis DM, Phda J et al. Phase I trial of O6-benzylguanine for patients under surgery for malignant glioma. J Clin Oncol 1998,16:3570-3575.
[40]潘强,杨学军.MGMT表达在胶质瘤对烷化剂耐药中的作用.国际神经病学神经外科学杂志.2009,36：33-36.
[41]Day RS 3rd, Ziolkowski CH, Scudiero DA, et al. Defective repair of alkylated DNA by human tumour and SV40-transformed human cell lines. Nature.1980, 288(5792):724-727.
[42]Esteller M, Hamilton SR, Burger PC, etal. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res.1999,59:793-797.
[43]Paus C, Murat A, Stupp R, et al. Role of MGMT and clinical applications in brain tumours. J Bull Cancer.2007,94:769-73.
[44]Balana C,Carrato C, Ramirez J, et al. Concordance and clinical value of MGMT promoter methylation pattern in tissue with paired serum and MGMT promoter methylation pattern in a series of glioblastoma(GB) patients.JClin Oncol,2008,26 Suppl:98.
[45]Kaicker S, McCrudden KW, Beck L, et al. Thalidomide is anti-angiogenic in a xenograft model of neuroblastoma. Int J Oncol.2003,23(6):1651-1655.
[46]Xiao YH, Yi H, Tan T, et al. Analysis of in vitro anti-leukemia effect of 5-aza-2'-deoxycitydine. Zhong Nan Da Xue Xue Bao Yi Xue Ban.2008, 33(4):344-352.
[47]Hart MG, Grant R, Garside R, et al. Temozolomide for high grade glioma. Cochrane Database Syst Rev.2008,8;(4):CD007415.
[48]Rush LJ, Raval A, Funchain P, et al. Epigenetic profiling in chronic lymphocytic leukemia reveals. Cancer Res,2004,64(7):2424-2433.
[49]Doom R, Zoutman WH, Dijkman R, et al. Epigenetic profiling of cutaneous T-cell lymphoma:promoter hypermethylation of multiple tumor suppressor genes including BCL7a, PTPRG, and p73. J Clin Oncol,2005,23(17):3886-3896.
[50]Toyota M, Issa JP. Methylated CpG island amplification for methylation analysis and cloning diferentially methylated sequences. Methods Mol Biol,2002,200: 101-110.
[51]Brena RM, Auer H, Kornacker K, et al. Quantification of DNA methylation in electrofluidics chips (Bio-COBRA). Nat Protoc,2006,1(1):52-58.
[52]Han W, Cauchi S, Herman JG, et al. DNA methylation mapping by tag-modified bisulfite genomic sequencing. Anal Biochem,2006,355(1):50-61.
[53]Weisenberger DJ, Campan M, Long TI, et al. Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Res,2005,33(21):6823-6836.
[54]Hou P, Shen JY, Ji MJ, et al. Microarray-based method for detecting methylation changes of p16(Ink4a) gene 5'-CpG islands in gastric carcinomas. World J Gastroenterol,2004,10(24):3553-3558.
[55]Gonzalgo ML, Liang G. Methylation-sensitive single-nucleotide primer extension(Ms-SNuPE) for quantitative measurement of DNA methylation. Nat Protoc,2007,2(8):1931-1936.
[56]Betz B, Florl AR, Seifert HH, et al. Denaturing high-performance liquid chromatography (DHPLC) as a reliable high-throughput prescreening method for aberrant promoter methylation in cancer. Hum Mutat,2004,23(6):612-620.
[57]Ueda S, Mineta T, Nakahara Y, et al. Induction of the DNA repair gene O6-methylguanine-DNA methyltransferase by dexamethasone in glioblastomas. J Neurosurg.2004,101(4):659-63.
[58]Chamberlain MC. Treatment options for gioblastoma. Neurosurg Focus.2006,20 (4) E2.
[1]Paus C, Murat A, Stupp R, et al. Role of MGMT and clinical applications in brain tumors. J Bull Cancer.2007,94(9):769-73.
[2]Bearzaatto A, S zadkowski M, Macpherson P, et al. Epigenetic regulation of the MGMT and hMSH6 DNA repair genes in cells resistant to methylating agents. Cancer Res,2000,60 (12):3262-3270.
[3]Ostrowski LE, von Wronski MA, Bigner SH, et al. Expression of O6-methylguanine-DNAmethyltransferase in malignant human glioma cell lines, Carcinogenesis,1991,12(9):1739-44.
[4]Hegi ME, Liu L, Herman JG, et al. Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategiesto modulate MGMT activity. J Clin Oncol.2008, 26(25):4189-99.
[5]Rodriguez FJ, Thibodeau SN, Jenkins RB, et al. MGMT immunohistochemical expression and promoter methylation in human glioblastoma. Appl Immunohistochem Mol Morphol.2008,16(1):59-65.
[6]Ma J, Murphy M, O'Dwyer PJ, et al. Biochemical changes associated with a multidrug-resistant phenotype of a human glioma cell line with temozolomide-acquired resistance. Biochem pharmacol 2002,63(7):1219-28.
[7]Casorelli I, Russo MT, Bignami M. Role of mismatch repair and MGMT in response to anticancer therapies. Anticancer Agents Med Chem.2008,8(4):368-80.
[8]Friedman H.S, McLendon R.E, Kerby T, et al. DNA mismatch-repair and O6-alkylguanine-DNA-alkyltransferase analysis and response to Temodal in newly diagnosed malignant glioma, J Clin Oncol.1998,16(12):3851-3857.
[9]林志雄,谭淑莲,周爱萍等.影响替莫唑胺治疗脑胶质瘤效果的非病例级别因素初步探讨.[J]中国现代神经疾病杂志,2008,8(5)：437-441.
[10]Froelich-Ammon SJ, Osheroff N. Topoisomerase poisons:harnessing the dark side of enzyme mechanism. J Biol Chem.1995,270(37):21429-32.
[11]Kuriyama M, T sutsui K, Tsutsui K, et al. Induction of resistance to etoposide and adriamycin in a human glioma cell line treated with antisense oligonucleotid complementary to the messenger ribonucleic acid of deoxyribonucleic acid topoisomerase Ⅱ alpha. Neurol Med Chir (Tokyo).1997,37(9):655-61; discussion 661-2.
[12]Uesaka T, Shono T, Kuga D, et al. Enhanced expression of DNA topoisomerase Ⅱ genes in human medulloblastoma and its possible association with etoposide sensitivity. J Neuro-oncol.2007,84(2):119-29. Epub 2007 Mar 15.
[13]Shi H, Lu D, Shu Y, et al. Expression of multidrug-resistance-related proteins P-glyco-protein, glutathione-S-transferases, topoisomerase-Ⅱ and lung resistance protein in primary gastric cardiac adenocarcinoma. Cancer Invest.2008, 26(4):344-51.
[14]Monzo M, Rosell R, Taron M. Drug resistance in non-small cell lung cancer [J]. Lung cancer,2001,34(Suppl 2):S91-94.
[15]Kong JX, Song Q, Liu YP, et al. Inhibition of multidrug resistance gene 1 expression in glioma by antisense oligodeoxynucleotides. Zhong guo Yi Xue Ke Xue Yuan Xue Bao.2005,27(2):211-5.
[16]Spiegl-Kreinecker S, Buchroithner J, Elbling L, et al. Expression and functional activity of the ABC-transporter proteins P-glycoprotein and multidrug-resistance protein 1 in human brain tumor cells and astrocytes. J Neurooncol.2002, 57(1):27-36.
[17]Bahr O, Rieger J, Duffner F, et al. P-glycoprotein and multidrug resistance-associated protein mediate specific patterns of multidrug resistance in malignant glioma cell lines, but not in primary glioma cells. Brain Pathol.2003, 13(4):482-94.
[18]Cole SP, Deeley RG. Multidrug resistance mediated by the ATP-binding cassette trans-porter protein MRP. Bioessays.1998,20(11):931-40.
[19]Deeley RG, Cole SP. Substrate recognition and transport by multidrug resistance protein 1 (ABCC1). FEBS Lett.2006,580(4):1103-11.
[20]Benyahia B, Huguet S, Decleves X, et al. Multidrug resistance-associated protein MRP1 expression in human gliomas:chemosensitization to vincristine and etoposide by indome-thacin in human glioma cell lines overexpressing MRP1. J Neurooncol.2004,66 (1-2):65-70.
[21]Nies AT, Jedlitschky G, Konig J, et al. Expression and immunolocalization of the multidrug resistance proteins, MRP1-MRP6 (ABCC1-ABCC6), in human brain. Neuroscience,2004,129:349-360.
[22]Calatozzolo C, Gelati M, Ciusani E, et al. Expression of drug resistance protein P-gp, MRP1, MRP3, MRP5 and GST-pi in human glioma.J Neurooncol,2005, 74:113-121.
[23]Pollack I F, Kwaecki S, Lazo J S. Blocking of glioma proliferation in vitro and in vivo and potentiating the effects of BCNU and cisplatin:UCN-01, a selective protein kinase Cinhibitor, J Neurosurg.1996,84(6):1024-1032.
[24]Doyle LA, Ross DD. Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene 2003,22:7340-7358.
[25]Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci USA.1998, 95:15665-15670.
[26]Zhang W, Mojsilovic-Petrovic J, Andrade MF, et al. The expression and functional characterization of ABCG2 in brain endothelial cells and vessels. FASEB J. 2003,17(14):2085-7.
[27]Aronica E, Gorter JA, Redeker S, et al. Localization of breast cancer resistance protein (BCRP) in microvessel endothelium of human control and epileptic brain. Epilepsia,2005,46:849-857.
[28]Sisodiya SM, Martinian L, Scheffer GL, et al. Major vault protein, a marker of drug resistance, is upregulated in refractory epilepsy. Epilepsia.2003, 44(11):1388-96.
[29]Sasaki T, Hankins GR, Helm GA. Major vault protein/lung resistance-related protein (MVP/LRP) expression in nervoussystem tumors. Brain Tumor Path,2002, 19:59-62.
[30]Berger W, Spiegl-Kreinecker S, Buchroithner J, et al. Overexpression of the human major vault protein in astrocytic brain tumor cells. Int J Cancer,2001, 94:377-382.
[31]Iwadate Y, Fujimoto S, Tagawa M, et al. Association of p53 gene muation with decreased chemosensitivity in human malignant gliomas. Int J Cancer. 1996,69(3):236-40.
[32]Fojo T, Bates S. Strategies for reversing drug resistance. Oncogene,2003, 22(47):7512.
[33]Koda M, Reszec J, Sulkowska M, et al. Expression of the insulin-like growth factor-I receptor and proapoptotic Bax and Bak proteins in human colorectal cancer. Ann NY Acad Sci,2004,1030:377-383.
[34]Tang R, Faussat AM, Majdak P, et al. Valproic acid inhibits proliferation and induces apoptosis in acute myeloid leukemia cells expressing P-gp and MRP1. leukemia,2004,18(7):1246
[35]Tyagi D, Sharma BS, Gupta SK, et al. Expression of Bcl2 proto-oncogene in primary tumors of the central nervous system. Neurol India,2002,50:290-294.
[36]Weller M, Malipiero U, Aguzzi A, et al. Protooncogene bcl-2 gene transfer abrogates Fas/APO-1 antibody-mediated apoptosis of human malignant glioma cells and confers resistance to chemotherapeutic drugs and therapeutic irradiation. J Clin Invest.1995,95(6):2633-43.
[37]Newcomb EW, Bhalla SK, Parrish CL, et al. bcl-2 protein expression in astrocytomas in relation to patient survival and p53 gene status. Acta Neuropathol 1997,94:369-375.
[38]Bonnet D, Dick JE. Human acute myeloid leukemia is organ-ized as a hierarchy that originates from a primitive hematopoi etic cell. Nat Med,1997,3(7):730-737.
[39]Challen GA, Little MH. A side order of stem cells:the SPphenotype. Stem Cells 2006,24:3-12
[40]Hirschmann-Jax C, Foster AE, Wulf GG, et al. A distinct "side population" of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci USA, 2004,101 (39):14228-14233.
[41]Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer,2006,5:67-71.
[42]Shervington A, Lu C. Expression of multidrug resistance genes in normal and cancer stem cells. Czncer Invest,2008,26:55-542.
[43]Bi CL, Fang JS, Chen FH, et al. Chemoresistance of CD133(+) tumor stem cells from human brain glioma. Zhong Nan Da Xue Xue Bao Yi Xue Ban.2007, 32(4):568-73
[44]Caircross JG, Ueki K, Zlatescu MC, et al. Specific genetic predictors of chemotherapeutic response and survival in patients with anaplastic oligodendrogliomas. J Natl Cancer Res,2003,9(4):1461-1468.
[45]Ito M, Wakabayashi T, Natsume A, et al. Genetically heterogeneous glioblastoma recurring with disappearance of lp/19q losses:case report. Neurosurgery.2007, 61(1):E168-9; discussion E169.
[46]Ino Y, Betensky RA, Zlatescu MC, et al. Molecular subtypes of anaplastic oligodendro-glioma:implications for patient management at diagnosis. Clin cancer Res,2001,7(4):839-845.
[47]Jenkins RB, Blair H, Ballman KV, et al. At (1;19) (q10;p10) Mediates the combined deletions of 1p and 19q and predicts a better prognosis of patients with oligodendroglioma. Cancer Res,2006,66:9852-61.
[48]Idbaih A, Omuro A, Ducray F, et al. Molecular genetic markers as predictors of response to chemotherapy in gliomas. Curr Opin Oncol.2007,19(6):606-11.
[2]潘强,杨学军,高松等.替莫唑胺改变人U251胶质瘤细胞系耐药特性机制的体外研究[J].中国现代神经疾病杂志,2009,9(6)：583-588.
[3]GAO Song, YANG Xue-jun, ZHANG Wen-gao, et al. Mechanism of thalidomide to enhance cytotoxicity of temozolomide in U251-MG glioma cells in vitro. Chin Med J,2009; 122 (11):1260-1266.
[4]Nicholas GA, Tracy TB. New Treatment Strategies for Malignant Gliomas. Oncologist.1999;4:209-224
[5]Mineura K, Yanagisawa T, Watanabe K, et al. Human brain tumor O6-methylaguanine-DNA methyltransferase mRNA and its significance as indicator of selective chloroethylnitrosourea chemotherapy. Int J Cancer.1996, 69:420-425.
[6]Paus C, Murat A, Stupp R,et al. Role of MGMT and clinical applications in brain tumors. J Bull Cancer.2007,94(9):769-73.
[7]Bearzaatto A,S zadkowski M, Macpherson P, et al. Epigenetic regulation of the MGMT and hMSH6 DNA repair genes in cells resistant to methylating agents. Cancer Res,2000,60 (12):3262-3270.
[8]Teo SK, Stirling DI, Zeldis JB. Thalidomide as a novel therapeutic agent:new uses for an old produt. Drug Discov Today.2005,10:104-107.
[9]Chang SM, Lamborn KR, Malec M, et al. Phase Ⅱ study of temozolomide and thalidomide with radiation therapy for newly diagnosed glioblastoma multiforme. Int J Radiat Oncol Biol Phys.2004,60(2):353-357.
[10]Puduvalli VK, Yung WK, Hess KR, et al.Phase Ⅱ study of fenretinide (NSC 374551) in adults with recurrent malignant gliomas:A North American Brain Tumor Consortium study. J Clin Oncol.2004,22(21):4282-4289.
[11]Baumann F, Bjeljac M, Kollias SS, et al. Combined thalidomide and temozolomide treatment in patients with glioblastoma multiforme. J Neurooncol.2004, 67(1-2):191-200.
[12]常洪波,章翔,甄海宁,等.白藜芦醇抑制SHG-44胶质瘤细胞生长实验研究 [J].中华神经外科疾病研究杂志,2006,5(2)：119-123.
[13]李晓兴,王之敏,左剑玲等.替莫唑胺诱导的人脑胶质瘤耐药细胞系的建立及其耐药性逆转.苏州大学学报(医学版)2007,27：169-171.
[14]Kanzawa T, Germano IM,Komata T,et al. Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ. 2004,11:448-457.
[15]Kim JD, Choi BK, Bae JS, et al. Clonin and characterization of GITR ligand [J]. Genes Immun,2003,4(8):564-569.
[16]王伯沄.免疫荧光组织化学和荧光组织化学技术。第四军医大学科技资料增刊,1974,2(1)：5.
[17]周庚寅主编.组织病理学技术[M].北京：北京大学医学出版社,2006：58.
[18]Markus B.Anticancer drug resistence in primary human brain tumors. Brain Res Rev.2001,35:161-204.
[19]Uesaka T, Shono T, Kuga D,et al. Enhanced expression of DNA topoisomerase Ⅱ genes in human medulloblastoma and its possible association with etoposide sensitivity. J Neuro-oncol.2007,84(2):119-29. Epub 2007 Mar 15.
[20]Monzo M, Rosell R, Taron M. Drug resistance in non-small cell lung cancer [J]. Lung cancer,2001,34(Suppl 2):S91-94.
[21]Spiegl-Kreinecker S, Buchroithner J, Elbling L, et al. Expression and functional activity of the ABC-transporter proteins P-glycoprotein and multidrug-resistance protein 1 in human brain tumor cells and astrocytes. J Neurooncol.2002, 57(1):27-36.
[22]Bahr O, Rieger J, Duffner F, et al. P-glycoprotein and multidrug resistance-associated protein mediate specific patterns of multidrug resistance in malignant glioma cell lines, but not in primary glioma cells. Brain Pathol. 2003,13(4):482-94.
[23]Cole SP, Deeley RG. Multidrug resistance mediated by the ATP-binding cassette trans-porter protein MRP. Bioessays.1998,20(11):931-40.
[24]Benyahia B, Huguet S, Decleves X, et al. Multidrug resistance-associated protein MRP1 expression in human gliomas:chemosensitization to vincristine and etoposide by indome-thacin in human glioma cell lines overexpressing MRP1. J Neurooncol.2004,66 (1-2):65-70.
[25]Sisodiya SM, Martinian L, Scheffer GL, et al. Major vault protein, a marker of drug resistance, is upregulated in refractory epilepsy. Epilepsia.2003, 44(11):1388-96.
[26]Sasaki T, Hankins GR, Helm GA. Major vault protein/lung resistance-related protein (MVP/LRP) expression in nervoussystem tumors. Brain Tumor Path 2002, 19:59-62.
[27]Pollack I F, Kwaecki S, Lazo J S. Blocking of glioma proliferation in vitro and in vivo and potentiating the effects of BCNU and cisplatin:UCN-01, a selective protein kinase Cinhibitor, J Neurosurg.1996,84(6):1024-1032.
[28]Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci USA.1998,95:15665-70.
[29]Zhang W, Mojsilovic-Petrovic J, Andrade MF, et al. The expression and functional characterization of ABCG2 in brain endothelial cells and vessels. FASEB J.2003, 17(14):2085-7. Epub 2003 Sep 4.
[30]Iwadate Y, Fujimoto S, Tagawa M, et al. Association of p53 gene muation with decreased chemosensitivity in human malignant gliomas. Int J Cancer.1996, 69(3):236-40.
[31]Fojo T, Bates S. Strategies for reversing drug resistance. Oncogene,2003, 22(47):7512.
[32]Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer,2006,5:67-71.
[33]Shervington A, Lu C. Expression of multidrug resistance genes in normal and cancer stem cells. Czncer Invest,2008,26:55-542.
[34]Bi CL, Fang JS, Chen FH, et al. Chemoresistance of CD133(+) tumor stem cells from human brain glioma. Zhong Nan Da Xue Xue Bao Yi Xue Ban.2007, 32(4):568-73.
[35]Caircross JG, Ueki K, Zlatescu MC, et al. Specific genetic predictors of chemotherapeutic response and survival in patients with anaplastic oligodendrogliomas. J Natl Cancer Res,2003,9(4):1461-1468.
[36]Ito M, Wakabayashi T, Natsume A, et al. Genetically heterogeneous glioblastoma recurring with disappearance of lp/19q losses:case report. Neurosurgery.2007, 61(1):E168-9; discussion E169.
[37]潘强,杨学军.胶质瘤化疗耐药分子机制研究进展.中国临床神经外科杂志,2010,15(2)：118-122.
[38]RI弗雷谢尼,动物细胞培养基本技术指南(分子克隆实验指南系列).第4版.北京：科学出版社,2004：398-400.
[39]Friedman HS, Kokkinakis DM, Phda J et al. Phase I trial of O6-benzylguanine for patients under surgery for malignant glioma. J Clin Oncol 1998,16:3570-3575.
[40]潘强,杨学军.MGMT表达在胶质瘤对烷化剂耐药中的作用.国际神经病学神经外科学杂志.2009,36：33-36.
[41]Day RS 3rd, Ziolkowski CH, Scudiero DA, et al. Defective repair of alkylated DNA by human tumour and SV40-transformed human cell lines. Nature.1980, 288(5792):724-727.
[42]Esteller M, Hamilton SR, Burger PC, etal. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res.1999,59:793-797.
[43]Paus C, Murat A, Stupp R, et al. Role of MGMT and clinical applications in brain tumours. J Bull Cancer.2007,94:769-73.
[44]Balana C,Carrato C, Ramirez J, et al. Concordance and clinical value of MGMT promoter methylation pattern in tissue with paired serum and MGMT promoter methylation pattern in a series of glioblastoma(GB) patients.JClin Oncol,2008,26 Suppl:98.
[45]Kaicker S, McCrudden KW, Beck L, et al. Thalidomide is anti-angiogenic in a xenograft model of neuroblastoma. Int J Oncol.2003,23(6):1651-1655.
[46]Xiao YH, Yi H, Tan T, et al. Analysis of in vitro anti-leukemia effect of 5-aza-2'-deoxycitydine. Zhong Nan Da Xue Xue Bao Yi Xue Ban.2008, 33(4):344-352.
[47]Hart MG, Grant R, Garside R, et al. Temozolomide for high grade glioma. Cochrane Database Syst Rev.2008,8;(4):CD007415.
[48]Rush LJ, Raval A, Funchain P, et al. Epigenetic profiling in chronic lymphocytic leukemia reveals. Cancer Res,2004,64(7):2424-2433.
[49]Doom R, Zoutman WH, Dijkman R, et al. Epigenetic profiling of cutaneous T-cell lymphoma:promoter hypermethylation of multiple tumor suppressor genes including BCL7a, PTPRG, and p73. J Clin Oncol,2005,23(17):3886-3896.
[50]Toyota M, Issa JP. Methylated CpG island amplification for methylation analysis and cloning diferentially methylated sequences. Methods Mol Biol,2002,200: 101-110.
[51]Brena RM, Auer H, Kornacker K, et al. Quantification of DNA methylation in electrofluidics chips (Bio-COBRA). Nat Protoc,2006,1(1):52-58.
[52]Han W, Cauchi S, Herman JG, et al. DNA methylation mapping by tag-modified bisulfite genomic sequencing. Anal Biochem,2006,355(1):50-61.
[53]Weisenberger DJ, Campan M, Long TI, et al. Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Res,2005,33(21):6823-6836.
[54]Hou P, Shen JY, Ji MJ, et al. Microarray-based method for detecting methylation changes of p16(Ink4a) gene 5'-CpG islands in gastric carcinomas. World J Gastroenterol,2004,10(24):3553-3558.
[55]Gonzalgo ML, Liang G. Methylation-sensitive single-nucleotide primer extension(Ms-SNuPE) for quantitative measurement of DNA methylation. Nat Protoc,2007,2(8):1931-1936.
[56]Betz B, Florl AR, Seifert HH, et al. Denaturing high-performance liquid chromatography (DHPLC) as a reliable high-throughput prescreening method for aberrant promoter methylation in cancer. Hum Mutat,2004,23(6):612-620.
[57]Ueda S, Mineta T, Nakahara Y, et al. Induction of the DNA repair gene O6-methylguanine-DNA methyltransferase by dexamethasone in glioblastomas. J Neurosurg.2004,101(4):659-63.
[58]Chamberlain MC. Treatment options for gioblastoma. Neurosurg Focus.2006,20 (4) E2.
[1]Paus C, Murat A, Stupp R, et al. Role of MGMT and clinical applications in brain tumors. J Bull Cancer.2007,94(9):769-73.
[2]Bearzaatto A, S zadkowski M, Macpherson P, et al. Epigenetic regulation of the MGMT and hMSH6 DNA repair genes in cells resistant to methylating agents. Cancer Res,2000,60 (12):3262-3270.
[3]Ostrowski LE, von Wronski MA, Bigner SH, et al. Expression of O6-methylguanine-DNAmethyltransferase in malignant human glioma cell lines, Carcinogenesis,1991,12(9):1739-44.
[4]Hegi ME, Liu L, Herman JG, et al. Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategiesto modulate MGMT activity. J Clin Oncol.2008, 26(25):4189-99.
[5]Rodriguez FJ, Thibodeau SN, Jenkins RB, et al. MGMT immunohistochemical expression and promoter methylation in human glioblastoma. Appl Immunohistochem Mol Morphol.2008,16(1):59-65.
[6]Ma J, Murphy M, O'Dwyer PJ, et al. Biochemical changes associated with a multidrug-resistant phenotype of a human glioma cell line with temozolomide-acquired resistance. Biochem pharmacol 2002,63(7):1219-28.
[7]Casorelli I, Russo MT, Bignami M. Role of mismatch repair and MGMT in response to anticancer therapies. Anticancer Agents Med Chem.2008,8(4):368-80.
[8]Friedman H.S, McLendon R.E, Kerby T, et al. DNA mismatch-repair and O6-alkylguanine-DNA-alkyltransferase analysis and response to Temodal in newly diagnosed malignant glioma, J Clin Oncol.1998,16(12):3851-3857.
[9]林志雄,谭淑莲,周爱萍等.影响替莫唑胺治疗脑胶质瘤效果的非病例级别因素初步探讨.[J]中国现代神经疾病杂志,2008,8(5)：437-441.
[10]Froelich-Ammon SJ, Osheroff N. Topoisomerase poisons:harnessing the dark side of enzyme mechanism. J Biol Chem.1995,270(37):21429-32.
[11]Kuriyama M, T sutsui K, Tsutsui K, et al. Induction of resistance to etoposide and adriamycin in a human glioma cell line treated with antisense oligonucleotid complementary to the messenger ribonucleic acid of deoxyribonucleic acid topoisomerase Ⅱ alpha. Neurol Med Chir (Tokyo).1997,37(9):655-61; discussion 661-2.
[12]Uesaka T, Shono T, Kuga D, et al. Enhanced expression of DNA topoisomerase Ⅱ genes in human medulloblastoma and its possible association with etoposide sensitivity. J Neuro-oncol.2007,84(2):119-29. Epub 2007 Mar 15.
[13]Shi H, Lu D, Shu Y, et al. Expression of multidrug-resistance-related proteins P-glyco-protein, glutathione-S-transferases, topoisomerase-Ⅱ and lung resistance protein in primary gastric cardiac adenocarcinoma. Cancer Invest.2008, 26(4):344-51.
[14]Monzo M, Rosell R, Taron M. Drug resistance in non-small cell lung cancer [J]. Lung cancer,2001,34(Suppl 2):S91-94.
[15]Kong JX, Song Q, Liu YP, et al. Inhibition of multidrug resistance gene 1 expression in glioma by antisense oligodeoxynucleotides. Zhong guo Yi Xue Ke Xue Yuan Xue Bao.2005,27(2):211-5.
[16]Spiegl-Kreinecker S, Buchroithner J, Elbling L, et al. Expression and functional activity of the ABC-transporter proteins P-glycoprotein and multidrug-resistance protein 1 in human brain tumor cells and astrocytes. J Neurooncol.2002, 57(1):27-36.
[17]Bahr O, Rieger J, Duffner F, et al. P-glycoprotein and multidrug resistance-associated protein mediate specific patterns of multidrug resistance in malignant glioma cell lines, but not in primary glioma cells. Brain Pathol.2003, 13(4):482-94.
[18]Cole SP, Deeley RG. Multidrug resistance mediated by the ATP-binding cassette trans-porter protein MRP. Bioessays.1998,20(11):931-40.
[19]Deeley RG, Cole SP. Substrate recognition and transport by multidrug resistance protein 1 (ABCC1). FEBS Lett.2006,580(4):1103-11.
[20]Benyahia B, Huguet S, Decleves X, et al. Multidrug resistance-associated protein MRP1 expression in human gliomas:chemosensitization to vincristine and etoposide by indome-thacin in human glioma cell lines overexpressing MRP1. J Neurooncol.2004,66 (1-2):65-70.
[21]Nies AT, Jedlitschky G, Konig J, et al. Expression and immunolocalization of the multidrug resistance proteins, MRP1-MRP6 (ABCC1-ABCC6), in human brain. Neuroscience,2004,129:349-360.
[22]Calatozzolo C, Gelati M, Ciusani E, et al. Expression of drug resistance protein P-gp, MRP1, MRP3, MRP5 and GST-pi in human glioma.J Neurooncol,2005, 74:113-121.
[23]Pollack I F, Kwaecki S, Lazo J S. Blocking of glioma proliferation in vitro and in vivo and potentiating the effects of BCNU and cisplatin:UCN-01, a selective protein kinase Cinhibitor, J Neurosurg.1996,84(6):1024-1032.
[24]Doyle LA, Ross DD. Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene 2003,22:7340-7358.
[25]Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci USA.1998, 95:15665-15670.
[26]Zhang W, Mojsilovic-Petrovic J, Andrade MF, et al. The expression and functional characterization of ABCG2 in brain endothelial cells and vessels. FASEB J. 2003,17(14):2085-7.
[27]Aronica E, Gorter JA, Redeker S, et al. Localization of breast cancer resistance protein (BCRP) in microvessel endothelium of human control and epileptic brain. Epilepsia,2005,46:849-857.
[28]Sisodiya SM, Martinian L, Scheffer GL, et al. Major vault protein, a marker of drug resistance, is upregulated in refractory epilepsy. Epilepsia.2003, 44(11):1388-96.
[29]Sasaki T, Hankins GR, Helm GA. Major vault protein/lung resistance-related protein (MVP/LRP) expression in nervoussystem tumors. Brain Tumor Path,2002, 19:59-62.
[30]Berger W, Spiegl-Kreinecker S, Buchroithner J, et al. Overexpression of the human major vault protein in astrocytic brain tumor cells. Int J Cancer,2001, 94:377-382.
[31]Iwadate Y, Fujimoto S, Tagawa M, et al. Association of p53 gene muation with decreased chemosensitivity in human malignant gliomas. Int J Cancer. 1996,69(3):236-40.
[32]Fojo T, Bates S. Strategies for reversing drug resistance. Oncogene,2003, 22(47):7512.
[33]Koda M, Reszec J, Sulkowska M, et al. Expression of the insulin-like growth factor-I receptor and proapoptotic Bax and Bak proteins in human colorectal cancer. Ann NY Acad Sci,2004,1030:377-383.
[34]Tang R, Faussat AM, Majdak P, et al. Valproic acid inhibits proliferation and induces apoptosis in acute myeloid leukemia cells expressing P-gp and MRP1. leukemia,2004,18(7):1246
[35]Tyagi D, Sharma BS, Gupta SK, et al. Expression of Bcl2 proto-oncogene in primary tumors of the central nervous system. Neurol India,2002,50:290-294.
[36]Weller M, Malipiero U, Aguzzi A, et al. Protooncogene bcl-2 gene transfer abrogates Fas/APO-1 antibody-mediated apoptosis of human malignant glioma cells and confers resistance to chemotherapeutic drugs and therapeutic irradiation. J Clin Invest.1995,95(6):2633-43.
[37]Newcomb EW, Bhalla SK, Parrish CL, et al. bcl-2 protein expression in astrocytomas in relation to patient survival and p53 gene status. Acta Neuropathol 1997,94:369-375.
[38]Bonnet D, Dick JE. Human acute myeloid leukemia is organ-ized as a hierarchy that originates from a primitive hematopoi etic cell. Nat Med,1997,3(7):730-737.
[39]Challen GA, Little MH. A side order of stem cells:the SPphenotype. Stem Cells 2006,24:3-12
[40]Hirschmann-Jax C, Foster AE, Wulf GG, et al. A distinct "side population" of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci USA, 2004,101 (39):14228-14233.
[41]Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer,2006,5:67-71.
[42]Shervington A, Lu C. Expression of multidrug resistance genes in normal and cancer stem cells. Czncer Invest,2008,26:55-542.
[43]Bi CL, Fang JS, Chen FH, et al. Chemoresistance of CD133(+) tumor stem cells from human brain glioma. Zhong Nan Da Xue Xue Bao Yi Xue Ban.2007, 32(4):568-73
[44]Caircross JG, Ueki K, Zlatescu MC, et al. Specific genetic predictors of chemotherapeutic response and survival in patients with anaplastic oligodendrogliomas. J Natl Cancer Res,2003,9(4):1461-1468.
[45]Ito M, Wakabayashi T, Natsume A, et al. Genetically heterogeneous glioblastoma recurring with disappearance of lp/19q losses:case report. Neurosurgery.2007, 61(1):E168-9; discussion E169.
[46]Ino Y, Betensky RA, Zlatescu MC, et al. Molecular subtypes of anaplastic oligodendro-glioma:implications for patient management at diagnosis. Clin cancer Res,2001,7(4):839-845.
[47]Jenkins RB, Blair H, Ballman KV, et al. At (1;19) (q10;p10) Mediates the combined deletions of 1p and 19q and predicts a better prognosis of patients with oligodendroglioma. Cancer Res,2006,66:9852-61.
[48]Idbaih A, Omuro A, Ducray F, et al. Molecular genetic markers as predictors of response to chemotherapy in gliomas. Curr Opin Oncol.2007,19(6):606-11.