阿霉素在小鼠体内诱导S—180细胞株抗药性的实验研究

利用ＢＡＢＬ／Ｃ小鼠，腹腔接种Ｓ－１８０瘤细胞，阿霉素腹腔注射治疗１５个周期培育传代，得到抗阿霉素的Ｓ－１８０细胞株，此抗药细胞对阿霉素的抗药性比亲本细胞提高６６倍，对典型的ＤＮＡ拓扑异构醇Ⅱ抑制剂ＶＰ１６抗药性增加９倍，经免疫组化进一步证实，抗药细胞显示多药抗药基因产物Ｐ－１７０糖蛋白过表达，流式萤光细胞仪测试结果也表明抗药细胞比亲本细胞排出药物能力提高８９倍，可以肯定这是ＭＤＲ基因过表达产物所     

体内培育S-180R阿霉素抗药细胞株遗传稳定性分子生物学证据

目的从分子水平阐明ＢＡＬＢ／ｃ小鼠体内阿霉素抗药细胞株Ｓ－１８０Ｒ遗传稳定性证据。方法采用流式细胞技术,ＤＮＡ、ＲＮＡ分子杂交和ＲＴ－ＰＣＲ对Ｓ－１８０Ｒ进行分析。结果Ｓ－１８０Ｒ抗药细胞排药能力比亲本细胞提高近１００倍,抗药细胞峰半峰宽与峰高比值由０．５６变为０．２３。Ｓ－１８０Ｒ抗药细胞ＤＮＡ出现多药耐药基因的显著扩增和转录,并可见拓扑异构酶基因的转录降低,但多药耐药相关蛋白基因转录未见增强。结论与两年前建系时相比,Ｓ－１８０Ｒ抗药细胞抗药倍数提高,抗药性更加均一,抗药机理是以多药耐药为主,拓扑异构酶Ⅱ为辅。Ｓ－１８０Ｒ小鼠腹水瘤抗阿霉素细胞株是理想的研究阿霉素抗药和抗药逆转剂的体内动物实验模型。     

人膀胱癌多药抗药性建立及逆转的实验研究

目的建立抗药细胞模型，研究维拉帕米和奎宁对膀胱癌抗药性的逆转及其机制。方法应用阿霉素（ＡＤＭ）大剂量短暂冲击结合低浓度递增法，建立抗药细胞模型，测定抗癌药物５０％抑制浓度和细胞内阿霉素浓度。结果获得了具有多药抗药性的ＢＩＵ－８７／ＡＤＭ细胞，该细胞对ＡＤＭ的敏感性下降了２１倍，同时对表柔比星（表阿霉素，ｅｐｉｒｕｂｉｃｉｎ）、长春新碱、依托泊甙也具有显著的交叉耐药性，对顺铂、丝裂霉素、氟尿嘧啶和甲氨蝶呤无抗药性。ＢＩＵ－８７／ＡＤＭ细胞在脱离ＡＤＭ诱导第８周后，其抗药性保持９０％以上。ＢＩＵ－８７／ＡＤＭ细胞内ＡＤＭ聚积量显著低于ＢＩＵ－８７细胞（Ｐ＜０．０５）；维拉帕米、奎宁能使ＢＩＵ－８７／ＡＤＭ细胞内ＡＤＭ聚积量增加（Ｐ＜０．０５），两者联合应用基本上可逆转ＢＩＵ－８７／ＡＤＭ细胞的抗药性。结论（１）ＢＩＵ－８７／ＡＤＭ细胞具有多药抗药性（ＭＤＲ）表型，且抗药性稳定。（２）细胞内ＡＤＭ聚积量减少是抗药细胞的重要变化，维拉帕米、奎宁能逆转ＢＩＵ－８７／ＡＤＭ细胞的抗药性，可能与增加细胞内ＡＤＭ聚积量有关。     

含MDR基因的人乳腺癌MCF 7/pZMDR多药耐药细胞株的构建

目的：肿瘤细胞的多药抗药性（ＭＤＲ）是化疗失败的主要原因之一，Ｐ－糖蛋白高表达是ＭＤＲ的主要机制，逆转ＭＤＲ成为肿瘤治疗亟待解决的问题。目前用于研究抗药性和筛选逆转抗药性药物的模型较少，本实验拟构建多药抗药性细胞株。方法：采用基因工程技术，重组ＭＤＲ１基因ｃＤＮＡ于逆转录病毒载体ｐＺＩＲ－ＮｅｏＳＶ（Ｘ）的克隆位点，并用脂染胺（ｌｉｐｏｆｅｃｔＡＭＩＮＥ）介导的ＤＮＡ转移技术，将其转入包装细胞ＰＡ３１７中，收集含病毒子的培养上清液感染对药物敏感的人乳癌细胞株ＭＣＦ－７，经筛选培养基筛选，ＰＣＲ、免疫组化及阿霉素在细胞内的分布等实验。结果：含ＭＤＲ基因的逆转录病毒载体ｐＺＭＤＲ的构建方法证明是正确的，ＭＤＲ１ｃＤＮＡ已整合在染色体基因组中并表达Ｐ－糖蛋白。结论：建立的ＭＣＦ－７／ｐＺＭＤＲ细胞为一特异表达Ｐ－糖蛋白的多药抗药性转基因细胞株     

含MDR基因的人乳腺癌MCF7／pZMR多药耐药细胞株的构建

目的：肿瘤细胞的多药抗药民生（ＭＤＲ）是化学失败的主要原因之定，Ｐ－糖蛋白高表达是ＭＤＲ的主要机制，逆转ＭＤＲ成为肿瘤治疗亟待的问题。目前用于研究抗经性和筛选逆转抗药性药物的模型较少，本实验拟构建多药抗药性细胞株。方法；采用基因工程技术，重组ＭＤＲ１基因ＣＤＮＡ于逆转录病毒载体ｐＺＩＲ－ＮｅｃＳＶ（Ｘ）的克隆位点，并用脂染胺介民的ＤＮＡ转移技术，将其转入包装细胞ＰＡ３１７中，收集含病毒子的培养上清     

P—糖蛋白过表达不能完全解释MDR细胞株K562／Dox的抗药性

本研究探讨ＭＤＲ细胞内药物积聚减少究竟决定其抗药性的多大比例。ＭＤＲ细胞Ｋ５６２／Ｄｏｘ来源于反复用Ｄｏｘ处理人红白血病细胞株Ｋ５６２细胞。经ＭＴＴ法测定，Ｋ５６２／Ｄｏｘ对长春新碱等７种抗癌药的抗性提高了１２００￣１１倍不等。Ｋ５６２／Ｄｏｘ可被抗人Ｐ－ｇｐ单抗ＪＳＢ－１标记，表明其有Ｐ－ｇｐ高表达。细胞内药物积聚试验显示，Ｋ５６２／Ｄｏｘ细胞内３种蒽环类药阿霉素、表阿霉素和柔红霉素的积聚均显著     

耐顺铂人肺腺癌细胞系A549DDP的建立及耐药机制

采用递增顺铂（ＤＤＰ）浓度的方法，体外连续培养建成一株耐ＤＤＰ的人肺腺癌细胞系Ａ（５４９）ＤＤＰ，耐ＤＤＰ为亲代Ａ（５４９）的２４．４倍。在无ＤＤＰ的培养基中培养５月余，其耐药性仍稳定。Ａ（５４９）ＤＤＰ细胞内谷胱甘肽（ＧＳＨ）水平显著高于亲代细胞（Ｐ＜０．０１）。ＢＳＯ耗竭细胞内ＧＳＨ后，Ａ（５４９）ＤＤＰ细胞对ＤＤＰ敏感性增加５倍，ＢＳＯ对亲代Ａ（５４９）细胞无增敏作用，Ａ（５４９）ＤＤＰ细胞ＧＳＴ酶同功酶ＧＳＴ—π含量较Ａ（５４９）高１．６倍，却无ＧＳＴ基因扩增，表明ＧＳＨ／ＧＳＴ解毒系统参与Ａ（５４９）ＤＤＰ耐药性的产生。实验结果亦示Ａ（５４９）ＤＤＰ与卡铂及氨甲喋呤间存在交叉耐药，而与易产生ＭＤＲ或ａｔＭＤＲ之ＡＤＭ、ＶＣＲ、ＶＰ－１６、ＶＭ－２６无交叉耐药，Ａ（５４９）细胞无Ｐ－糖蛋白（Ｐ－ｇｐ）表达，Ｓｏｕｔｈｅｒｎｂｌｏｔ研究Ａ（５４９）ＤＤＰ无ｍｄｒｌＴｏｐｏⅡ基因扩增，提示Ａ（４５９）ＤＤＰ细胞系与ＭＤＲ及ａｔ－ＭＤＲ无交叉耐药。     

异搏定逆转人肺腺癌A_（549）的多药抗药性

作者以抗阿霉素（ＡＤＭ）的人肺腺癌Ａ５４９亚细胞系Ａ５４９／Ｒ１和Ａ５４９／Ｒ２为模型，对异搏定（ＶＰＬ）的体外逆转抗药性效果及其作用机理进行了初步探索。发现ＶＰＬ浓度在１０μｇ／ｍｌ以下时无直接抑癌作用；以１０μｇ／ｍｌ剂量ＶＰＬ与ＡＤＭ联合，能使Ａ５４９／Ｒ２细胞内ＡＤＭ聚积量显著增加（Ｐ＜０．０５），并能使抗药细胞的抗药指数（ＲＦ）显著下降。结果表明，ＶＰＬ在体外能有效地逆转肿瘤细胞的抗药性，此作用与ＶＰＬ同Ｐ－糖蛋白竞争性结合，阻止药物外排有关，而与其钙通道阻断作用无关。     

肝癌多药耐药细胞株P—gp,MRP,GST—π表达水平的观察

为研究人肝癌多药耐药细胞株的耐药机理，应用ＢＥＬ－７４０２细胞株，通过不断提高培养液中阿霉素（Ｄｏｘｏｒｕｂｉｃｉｎ）的浓度，长期筛选培养，得到肝癌多药耐药株ＢＥＬ－７４０２／Ｄｏｘ。经ＭＴＴ法检测ＢＥＬ－７４０２对长春新碱（ＶＣＲ）等８种抗癌药的抗性提高了２７倍～１１００倍。采用流式细胞技术检测了细胞株表面ＭＤＲ１蛋白Ｐ－ｇｐ、多药耐药相关蛋白ＭＲＰ及谷脱甘肽硫转移酶ＧＳＴ－π的表达；用ＲＴ－ＰＣＲ方法检测了ＭＤＲ及ＭＲＰ基因表达水平。流式细胞分析发现，９３．５％～９７．４％的ＢＥＬ－７４０２／Ｄｏｘ细胞表面Ｐ－ｇｐ表达阳性；８４．７％～９０．２％的ＢＥＬ－７４０２／Ｄｏｘ细胞表面ＭＲＰ表达阳性；ＢＥＬ－７４０２细胞与ＢＥＬ－７４０２／Ｄｏｘ细胞ＧＳＴ－π的表达无明显变化。ＲＴ－ＰＣＲ证实此细胞株中有ＭＤＲ及ＭＲＰｍＲＮＡ的较高表达。     

半乳糖基修饰的反义硫代寡核苷酸对肝癌多药耐药细胞株MDR_1基因过度表达的抑制作用

设计合成三种互补ＭＤＲ１ｍＲＮＡ的反义硫代寡核苷酸（ＡＳＯＤＮ），分别互补ＭＤＲ１ｍＲＮＡ序列起始区、含ＡＵＧ起始密码子区及针对Ｌｏｏｐ形成位点区的序列，作用于阿霉素（ＤＯＸ）诱导的肝癌多药耐药细胞株ＢＥＬ－７４０２／ＤＯＸ。经流式细胞分析及ＲＴ－ＰＣＲ的方法检测，发现三种ＡＳＯＤＮ均能不同程度地降低ＢＥＬ－７４０２／ＤＯＸ细胞膜表面Ｐ－ＧＰ含量；同时ＭＤＲ１ｍＲＮＡ的表达也有轻度降低，其中尤以互补ＭＤＲ１ｍＲ－ＮＡＬｏｏｐ形成位点的ＡＳＯＤＮ抑制作用最强。以导向配体Ｇａｌ１０－ＰＬＬ修饰此ＡＳＯＤＮ后，作用于ＢＥＬ－７４０２／ＤＯＸ细胞，发现与相同剂量未修饰ＡＳＯＤＮ比较，Ｐ－ＧＰ含量由７６．８％降至１９．８％；对ＡＤＭ的ＩＣ５０由１．２８μｇ／ｍｌ降至０．４２μｇ／ｍｌ。实验说明，半乳糖基修饰的互补ＭＤＲ１ｍＲＮＡＬｏｏｐ形成位点的ＡＳＯＤＮ能够明显降低肝癌多药耐药细胞膜表面Ｐ－ＧＰ的含量，并较大程度地逆转多药耐药细胞ＢＥＬ－７４０２／ＤＯＸ对化疗药物的耐受性。     

DEVELOPMENT OF AN ADRIAMYCIN RESISTANT MURINE TUMOR S-180 CELL LINE IN VIVO

Adriamycin resistant cells were obtained from low dotage treated BABL/c mice Inoculated with S-180 cells. Resistance of these cells for adriamycin was 66-fold more than their parental cells. The resistance for a typical DNA topoisomerase Ⅱ inhibitor VP16 (Etopcaide) was increased 9 times. Overexpression of multidrug resistant gene (MDR gene) products, P-glycoproteins (P-1 70), was also demonstrated by immunohistochemistry. Furthermore, the ability of the resistant cells to reduce net cellular drug accumulation measured by flow fluorescence cytometry was 89-fold higher than their parental cells. These results support the hypothesis that the resistance of S-180R cells to adriamycin was mainly due to the overexpression of P-glycoproteins. The S-180R cells will be useful to select drugs or some other therapeutic strategies to overcome multidrug resistance in vivo.     

INDUCTION OF APOPTOSIS IN S-180 AND S-180R TUMOR CELLS BY ADRIAMYCIN IN VIVO

Apoptosis of tumor cells have become a newstandard for chemotherapy. It is useful to demonstrateinduction of apoptosis in tumor cells by anti-cancer drugsin vivo. We reported the results of apoptosis induction inmurine tumor cell line S-180 and it’s resistant cell linc S-180R by adriamycin in different dose and different time.We found that apoptosis in S-180 cells could be inducedby low dose of adriamycin, the apoptosis was started at 24h. after the administration, and reached to 62.5% of thecells to apptosis until 72 h. Comparison with theparental cell line, only 13% of S-180R cells wereapoptosed. At high dose, 20% of S-180R cells wereapoptosed, whereas, almost all S-180 cells were killed inthe same time. The lymphocytes were appeared inabdominal cavity of the mice after treatment ofadriamycin for 24 h. It was very interested to find outthat there was no lymphocyte left in the abdominal cavityof the mice with S-180R cells treated at high dose ofadriamycin.     

Induction of apoptosis in S-180 and S-180R tumor cells by adriamycinin vivo

Apoptosis of tumor cells have become a new standard for chemotherapy. It is useful to demonstrate induction of apoptosis in tumor cells by anti-cancer drugsin vivo. We reported the results of apoptosis induction in murine tumor cell line S-180 and it’s resistant cell line S-180R by adriamycin in different dose and different time. We found that apoptosis in S-180 cells could be induced by low dose of adriamycin, the apoptosis was started at 24 h. after the administration, and reached to 62.5% of the cells to apptosis until 72 h. Comparison with the parental cell line, only 13% of S-180R cells were apoptosed. At high dose, 20% of S-180R cells were apoptosed, whereas, almost all S-180 cells were killed in the same time. The lymphocytes were appeared in abdominal cavity of the mice after treatment of adriamycin for 24 h. It was very interested to find out that there was no lymphocyte left in the abdominal cavity of the mice with S-180R cells treated at high dose of adriamycin.     

Characterisation of a mouse tumour cell line with in vitro derived resistance to verapamil

We have established a subline (EMT6/VRP) of the mouse tumour cell line EMT6/P with acquired resistance to the calcium transport blocker verapamil (VRP). The subline was 4-fold resistant to the cytoxicity of VRP alone compared with the parent line but of similar sensitivity to adriamycin, vincristine or colchicine. EMT6/VRP cells growing in 75 micrograms ml-1 VRP were morphologically different from and larger in diameter than EMT6/P cells, but these two parameters reverted almost to normal within 3 days of VRP removal, although resistance was retained. Expression of an mRNA coding for P-glycoprotein was similar in EMT6/VRP and the parent cell line, although considerable hyperexpression was seen in a multidrug resistant subline, EMT6/AR1.0. Cellular accumulation of both 3H-daunorubicin and 3H-VRP were greater in EMT6/VRP than in the parent line. Sensitisation to adriamycin by 3.3 micrograms ml-1 VRP was, however, somewhat reduced in EMT6/VRP (i.e. to 6.1-fold) compared with the 11-fold sensitisation seen in the parent line. It is clear that resistance to VRP seen in this cell line occurs via a different mechanism from the resistance to drugs such as adriamycin, vincristine and colchicine seen in multidrug resistant cell lines.     

Molecular biological evidences for the genetic stability of doxorubicin resistant cell line S-180Rin vivo

Objective: In order to assess the genetic stability of doxorubicin resistance sarcoma S-180R cell linein vivo. Methods: The drug resistant genes and molecules were examined by flow cytometry, Southern blot, Northern blot and RT-PCR. Results: The results showed that drug-efflux in S-180R increased nearly 100-folds, as compared with its parent cells, the rate of half peak width resistant cell/peak high decreased from 0.56 to 0.23 measured by flow cytometry after two years. The mdr1 gene amplified and overexpressed significantly in S-180R and the expression of topoisomerase II α gene decreased remarkably in S-180R. There was no significant different of the MRP expression between S-180R and S-180. Conclusion: These results indicated that drug resistance of S-180R was maintained and also increased. The major mechanism of drug resistance is the amplification and overexpression of mdr1 gene, the decreased expression of topoisomerase II α also contributed to it. So, S-180R is an ideal experimental model for the study of doxorubicin resistance and its reversionin vivo.     

人肝癌移植瘤多药耐药模型的建立及耐药机制的探讨

背景与目的肿瘤多药耐药是肿瘤化疗的主要障碍和研究热点,本研究拟建立人肝癌裸小鼠皮下及肝原位移植多药耐药模型,探讨其生物学特性和耐药机制的异同,为研究体内逆转肿瘤多药耐药提供理想的动物模型。方法人肝癌细胞系HepG2裸鼠肝原位、皮下移植后,用阿霉素腹腔注射诱导耐药。MTT法检测耐药细胞对抗肿瘤药的敏感性,流式细胞仪检测癌细胞膜蛋白P-糖蛋白(P-gp)、多药耐药相关蛋白(MRP)、肺耐药蛋白(LRP)的表达和细胞对罗丹明(R123)的外排作用,逆转录PCR检测耐药细胞中三种膜蛋白mRNA的表达。结果肝原位和皮下移植瘤耐药细胞形态及生物学行为符合人肝癌特征;对多种药物产生较明显的耐药性,其中对阿霉素的耐药倍数分别为26.4和24.6;肝皮下移植和原位移植组耐药细胞膜蛋白P-gp、MRP和LRP的阳性率均增高,分别为(77.3±2.1)%和(78.1±1.9)%,(72.1±4.3)%和(72.7±5.1)%,(31.1±1.0)%和(32.2±1.4)%。多药耐药基因的阳性率也明显增高,细胞对R123的外排作用增强;肝原位移植瘤组与皮下移植瘤组比较,多药耐药性差异没有统计学意义(P>0.05)。结论所建立的裸鼠肝原位及皮下移植瘤多药耐药模型符合人肿瘤多药耐药特征,为研究体内逆转多药耐药提供了理想的动物模型。     

Radiation and heat sensitivity of human T-lineage acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) clones displaying multiple drug resistance (MDR).

The hyperthermia as well as radiation responses of multidrug resistant (CEM/VLB100 with classical MDR and CEM/VM-1 with atypical MDR), methotrexate resistant (CEM/MTX) subclones of CCRF-CEM T-lineage ALL cell line were compared with those of a drug sensitive (CEM-1-3) subclone from the same parent cell line. Also analyzed were the hyperthermia as well as radiation responses of multidrug resistant (HL60/AR) and drug sensitive subclones of the HL60 AML cell line. Notably, the drug resistant subclones of CEM and HL60 were as sensitive to hyperthermia as were the drug sensitive subclones. Importantly, no thermotolerant plateau was observed in the hyperthermia survival curves of the drug resistant subclones, indicating that drug/multidrug resistance is not associated with a greater likelihood of thermal tolerance development during hyperthermia. Similarly, the drug resistant CEM and HL60 subclones were not more radiation resistant than the drug sensitive subclones. Thus, the classical or atypical forms of multidrug resistance or methotrexate resistance of the analyzed leukemic cell lines were not associated with radiation resistance. Furthermore, the radiation survival curves of the drug resistant subclones lacked a distinct initial shoulder and their n values were not greater than those of the drug sensitive subclones, suggesting that multidrug resistance is not associated with an increased ability to repair or accumulate sublethal radiation damage. Our findings provide evidence that there is no apparent association between drug/multidrug resistance and heat or radiation sensitivity of CEM T-lineage ALL or HL60 AML leukemia cells. The results of this study indicate that acquired resistance to methotrexate, vinblastine, vincristine, etoposide, actinomycin-D, adriamycin, or daunomycin, or pleiotropic multidrug resistance do not necessarily confer radiation resistance for human leukemic cells.     

Induction of in vitro resistance to 4'-epidoxorubicin and cis-dichlorodiammineplatinum in hepatoma cells.

We have developed in vitro resistance to 4'-epidoxorubicin (Epi-A) and cis-dichlorodiammineplatinum (cis-DDP) in one rat (MH1C1) and one human hepatoma cell line (HepG2). When compared to their parental cells, the Epi-A resistant rat cells were 17 times and the resistant human cells 27 times more resistant to Epi-A in terms of GI50 in the cell growth inhibition assay. The cis-DDP resistant rat cells were 20 times and the resistant human cells 12 times more resistant to cis-DDP. Cross-resistance to cis-DDP was observed in the Epi-A resistant rat cells but not in the human cells. The multidrug resistant gene product, GP 170, was markedly expressed in both Epi-A resistant substrains compared with their parent lines, suggesting a role of this protein in the development of resistance to Epi-A. Cadmium-binding proteins of metallothionein (MT) size bound 52% of cytosolic 109cadmium in the cis-DDP resistant human cells compared with 8% in the parental cells. This may indicate that these proteins contribute to the observed cis-DDP resistance.     

Sensitivity of multidrug resistant KB-C1 cells to an antibody-dextran-adriamycin conjugate

Resistance to adriamycin is an important limitation to the use of the drug in cancer therapy. This resistance is often a manifestation of the multidrug resistance phenotype. Studies with multidrug resistant cell lines in vitro may be useful to design approaches for overcoming the drug resistance encountered clinically. We investigated the possibility of overcoming adriamycin resistance in vitro in a multidrug resistant subline (KB-C1) of human epidermal carcinoma (KB-3-1) cells using antibody-mediated drug targeting. Adriamycin was conjugated through a dextran bridge to a monoclonal antibody (mAb), 10B, which bound to KB-3-1 cells with a Ka of 4 x 10(8) M-1. The conjugate retained immunoreactivity with the target cells. Adriamycin (0.2 micrograms/ml) caused a 50% inhibition of DNA synthesis in KB-3-1 cells, but failed to achieve this degree of inhibition in KB-C1 cells at levels as high as 10 micrograms/ml. In contrast, the 10B-dextran-adriamycin conjugate produced 50% inhibition of DNA synthesis in KB-C1 cells at a concentration of 2.5 micrograms/ml. This was significantly more cytotoxic than adriamycin conjugated to control mAb or bovine serum albumin (BSA). Similarly, a 10B-recombinant ricin A (rRA) immunotoxin was more cytotoxic to KB-C1 cells than free rRA. These results indicate that adriamycin resistance in KB-C1 cells in vitro can be partially overcome by specifically targeting adriamycin to the cells using an 10B-dextran-adriamycin conjugate. This approach may be useful in overcoming adriamycin resistance encountered during the course of cancer therapy.