An immunotoxin cytotoxic for breast cancer cells in vitro

A potent immunotoxin was formed by conjugating the murine monoclonal antibody 323/A3 to the A chain of ricin. The 323/A3 antibody recognizes an antigen expressed by most human breast cancers. When binding of 323/A3 is examined by enzyme-linked immunosorption assay, three human breast cell lines displayed strong binding, whereas two human breast cell lines and three non-breast cell lines displayed little or no binding. When the cell lines were tested at a concentration of 0.1 microgram/ml, those cell lines which displayed an abundance of antigen by enzyme-linked immunosorption assay were most sensitive to the effects of the 323/A3 immunotoxin. On the other hand, cell lines which displayed little or no antigen by enzyme-linked immunosorption assay were not inhibited by the immunotoxin at this concentration. Further examination of the effects of immunotoxin concentration on protein synthesis confirmed the sensitivity of those cell lines rich in the 323/A3 antigen over a broad dose range. Similarly, three cell lines which displayed little of the 323/A3 antigen demonstrated little inhibition of protein synthesis with various concentrations of 323/A3 immunotoxin. However, two cell lines which displayed little antigen were intermediate in their sensitivity to the 323/A3 immunotoxin. Preclinical evaluation of immunotoxins as potential therapeutic agents will require accurate and sensitive screening of a wide variety of cell types. The 323/A3 remains of interest in studying the effects of immunotoxin in a defined in vivo model system.     

Monovalent immunotoxin containing truncated form of Pseudomonas exotoxin as potent antitumor agent.

Recombinant truncated forms of Pseudomonas exotoxin A that lack the cell binding domain of Pseudomonas exotoxin A were coupled to an F(ab') fragment of a monoclonal antibody HB21 directed against the human transferrin receptor. One of these was NlysPE40. The other, NlysPE38QQR, has two amino groups on residues near the NH2-terminus and has no amino groups near the COOH-terminus. The proteins were linked by a stable thioether bond that connected the sulfhydryl group present in the hinge region of the antibody fragment to an amino group on the toxin. The F(ab')-PE40 immunotoxin, containing NlysPE40, exhibited potent cytotoxic activity on human carcinoma cell lines with a concentration of immunotoxin at which isotope incorporation falls by 50% when compared to nontreated cells (ID50) of 5.3 pM (0.5 ng/ml) on both the epidermoid carcinoma A431 and on the colon carcinoma Colo205. Immunotoxins made with whole antibody were considerably less active, with an ID50 of 15.9 pM (3.1 ng/ml) on these cell lines. F(ab')-PE38QQR, the immunotoxin containing NlysPE38QQR, was found to be the most active agent with an ID50 of 1.05 pM (0.1 ng/ml) on A431 cells. The greater cytotoxicity of immunotoxins containing fragmented antibody was probably due to the higher binding affinity of F(ab') conjugates in comparison to whole antibody conjugates to the transferrin receptor. The increase in cytotoxic activity of the immunotoxin made with NlysPE38QQR than that with NlysPE40 may reflect selective coupling of the toxin through NH2-terminal amino groups. The monovalent and divalent immunotoxins had dose-dependent antitumor effects on human epidermoid carcinoma xenografts in nude mice. A431 tumors completely regressed in all animals at a total dose of 105 pmol (10 micrograms) of F(ab')-PE38QQR and of 154 pmol (30 micrograms) of IgG-PE38QQR. Furthermore, the F(ab') immunotoxin was less toxic to mice than the conjugate containing IgG (840 pmol or 80 micrograms of total dose causing measurable adverse effects versus 208 pmol or 40 micrograms, respectively). Thus, a truncated Pseudomonas exotoxin A molecule coupled to the F(ab') fragment of an antibody is more active and less toxic in mice than an immunotoxin made with a whole antibody. Therefore, the therapeutic index for the monovalent immunotoxin is about four times better than that for the divalent immunotoxin.     

卡维地洛逆转膀胱癌细胞株多药耐药性

目的：研究卡维地洛对人膀胱癌耐药细胞株BIU-87／ADM的多药耐药性逆转作用。方法：应用MTT法检测人膀胱癌耐药细胞株(BIU-87／ADM)的耐药性；将卡维地洛与多柔比星联合作用于B10-87和BIU-87／ADM细胞，检测细胞内多柔比星聚集量。结果：卡维地洛与多柔比星共同温育BIU-87／ADM细胞后．能使BIU-87／ADM细胞内多柔比星聚积量增加，与单用多柔比星比较，差异有显著性。结论：卡维地洛对多柔比星有增敏作用，卡维地洛可逆转人膀胱癌细胞株BIU-87／ADM的多药耐药性。     

小鼠uroplakinⅡ启动子在人细胞中的作用

目的　研究小鼠UroplakinⅡ (UPⅡ )启动子在人细胞中的作用。方法　采用半定量RT PCR方法 ,检测人类膀胱移行细胞癌细胞系 (BIU 87)、肾癌细胞系 (GRC 1)、脐静脉血管内皮细胞系(EC)、肺癌细胞系 (A549)和皮肤成纤维细胞系 (Hs2 7)中UPⅡ基因mRNA的表达情况 ;同时构建pEGFP UPⅡ、pGL UPⅡ重组质粒 ,应用脂质体转染技术 ,将重组质粒转染入BIU 87、GRC 1、EC、A549和Hs2 7;利用共聚焦显微镜、流式细胞仪和微板检测仪观察细胞表达绿色荧光蛋白 (GFP)和荧光素酶(Luc)的情况。结果　UPⅡ基因mRNA在BIU 87中有较高的表达 ,而在其他组织细胞中表达极低。BIU 87表达GFP较多 (5～ 10 /HP) ,亮度较强 ,流式细胞仪测定表达量为 10 .1% ;而GRC 1、EC细胞表达GFP极少 (0～ 2 /HP) ,亮度暗 ,流式细胞仪测定表达量分别为 0 %和 1.8%。Luc在BIU 87中的表达量是其他组织细胞的 1.8～ 8.2倍 ,差异有显著性 (P <0 .0 1)。结论　UPⅡ特异性的表达在膀胱癌细胞中。小鼠的UroplakinⅡ启动子在人类细胞中仍具有膀胱特异性的启动活性     

膀胱癌雌激素受体表达与他莫昔芬拮抗治疗的研究

背景与目的： 探讨他莫昔芬(TAM)联合阿霉素(ADM)对膀胱癌BIU-87细胞株生长的抑制作用。 材料与方法： 采用免疫组织化学法检测雌激素受体的表达;应用MTT法测定不同浓度的TAM(1、5、10 μmol/L)及ADM(0.1、1、10 μmol/L),单独和联合作用对BIU-87细胞的生长抑制率,观察TAM的增敏作用;采用原位杂交法检测各实验组bcl-2 mRNA的水平。 结果： 膀胱癌BIU-87细胞表达雌激素受体;TAM单独作用在低浓度时(1 μmol/L)对BIU-87生长无抑制作用;TAM联合ADM作用时,对BIU-87细胞生长的抑制率增加,其中TAM 10 μmol/L组增加显著(P<0.05),显示TAM能提高ADM的敏感性,起到化疗的增敏作用;随着TAM浓度的增加和时间的延长,BIU-87细胞bcl-2 mRNA的表达下调。 结论： 他莫昔芬能增加膀胱癌细胞株BIU-87对化疗药物ADM的敏感性,并抑制细胞增殖;其增敏的作用机制可能与bcl-2的表达下调有关。     

三氧化二砷对膀胱癌细胞BIU-87耐药相关基因的影响

目的：探讨三氧化二砷（arsenic trioxide,As2O3）对耐羟基喜树碱的膀胱癌细胞株BIU-87/HCPT耐药性逆转的机制。方法：用As2O3与羟基喜树碱联合处理BIU-87/HCPT细胞后,用四甲基偶氮唑蓝法（MTT）检测细胞增殖,流式细胞仪检测细胞周期、细胞凋亡、P-gp、MRP1、GST-π和生存素（survivin）表达的变化。结果：As2O3与羟基喜树碱联合处理耐药膀胱移行细胞癌株BIU-87/HCPT细胞后,细胞生长受到明显抑制。单独应用As2O3的凋亡率为11.07%,As2O3联合羟基喜树碱后的凋亡率为18.23%,凋亡细胞百分比较对照组明显增多,As2O3明显增加羟基喜树碱的作用（P〈0.05）。As2O3使P-gp、MRP 1、GST-π和sur-vivin的表达明显减少,且均具有浓度依赖性（P〈0.05）。结论：As2 O3可减少耐药相关基因的表达,部分逆转膀胱癌细胞的耐药性,促进凋亡,且呈现浓度依赖性。     

三氧化二砷对膀胱癌细胞BIU-87耐药相关基因的影响

目的:探讨三氧化二砷(arsenic trioxide,As2O3)对耐羟基喜树碱的膀胱癌细胞株BIU-87/HCPT耐药性逆转的机制.方法:用As2O3与羟基喜树碱联合处理BIU-87/HCPT细胞后,用四甲基偶氮唑蓝法(MTT)检测细胞增殖,流式细胞仪检测细胞周期、细胞凋亡、P-gp、MRP1、GST-π和生存素(survivin)表达的变化.结果:As2O3与羟基喜树碱联合处理耐药膀胱移行细胞癌株BIU-87/HCPT 细胞后,细胞生长受到明显抑制.单独应用As2O3的凋亡率为11.07%,As2O3联合羟基喜树碱后的凋亡率为18.23%,凋亡细胞百分比较对照组明显增多,As2O3明显增加羟基喜树碱的作用(P<0.05).As2O3使P-gp、MRP 1、GST-π和survivin的表达明显减少,且均具有浓度依赖性(P<0.05).结论:As2O3可减少耐药相关基因的表达,部分逆转膀胱癌细胞的耐药性,促进凋亡,且呈现浓度依赖性.     

As2O3对人膀胱癌细胞株BIU-87作用的体外研究

目的　观察三氧化二砷 (As2 O3 )对人膀胱癌细胞株BIU 87的作用 ,并探讨其作用机制。方法　采用四氮唑蓝(MTT )法检测As2 O3 不同浓度、不同作用时间对BIU 87细胞株的生长抑制率 ;流式细胞术 (FCM )检测不同浓度As2 O3 作用 72h后细胞中凋亡相关蛋白Fas、bcl 2的表达及细胞周期变化。结果　As2 O3 可有效抑制BIU 87细胞株的生长 ,具有浓度、时间依赖性特点。Fas、bcl 2的表达与As2 O3 浓度增高分别呈正、负相关 (P <0 .0 5 ) ,且随As2 O3 浓度增高Fas、bcl 2的表达呈负相关 (P <0 .0 5 )。随As2 O3 浓度增高细胞周期被阻滞在G0 /G1期。结论　As2 O3 可有效抑制人膀胱癌BIU 87细胞株的生长增殖 ,阻滞细胞周期及诱导细胞凋亡可能起了重要作用     

Anti-transferrin receptor antibody linked to Pseudomonas exotoxin as a model immunotoxin in human ovarian carcinoma cell lines

The present in vitro study was performed to evaluate the potential usefulness of immunotoxins in treating human ovarian carcinomas. A monoclonal antibody against the human transferrin receptor was covalently linked to Pseudomonas exotoxin. The activity of this immunotoxin (anti-TFR-PE) was studied in five ovarian carcinoma cell lines, a breast carcinoma cell line (MCF-7), and in KB cells. The ovarian carcinoma cell lines included one previously established cell line (A1847) and four recent isolates obtained from the malignant ascites of patients with metastatic ovarian carcinoma (OVCAR cell lines). While all cell lines showed inhibition of protein synthesis by anti-TFR-PE, there were quantitative differences when the level of protein synthesis was assayed after a 12-hr incubation with the immunotoxin. These differences resulted from different kinetics of anti-TFR-PE activity in the various cell lines. Higher levels of cellular binding and internalization of anti-TFR were shown to contribute to increased toxicity of anti-TFR-PE. Verapamil increased the rate of protein synthesis inhibition and thus enhanced the toxicity of anti-TFR-PE in the OVCAR cell lines.     

三氧化二砷对膀胱癌BIU-87细胞增殖及生存素表达的影响

目的：探讨三氧化二砷（arsenic trioxide,As2O3）对膀胱癌BIU-87细胞增殖及生存素表达的影响。方法：用As2O3处理膀胱移行细胞癌株BIU-87后,用四甲基偶氮唑蓝法（MTT）检测细胞增殖;用细胞流式仪检测细胞周期及凋亡,生存素（Survivin）表达的变化。结果：三氧化二砷作用BIU-87细胞后,细胞生长受到明显的抑制,Survivin的表达明显减少,凋亡细胞百分比较对照组明显增多,且均具有浓度依赖性。结论：As2O3对人膀胱癌细胞株BIU-87有抑制其生长、增殖和诱导凋亡的作用,且呈现浓度依赖性。