重组腺病毒介导人野生型p53,GM-CSF和B7-1基因对肿瘤化疗耐药细胞生物学行为的影响

目的：探讨重组腺病毒介导人野生型p５３,ＧＭ－ＣＳＦ和Ｂ７－１基因对肿瘤化疗耐药 细胞生物学行为的影响,为临床使用该重组腺病毒治疗多药耐药的肿瘤提供实验基础。方法：选用p５３异常的ＫＢ-s（ＶＣＲ敏感）和ＫＢv２００（ＶＣＲ耐药）细胞作为肿瘤化疗药物敏感和耐药的模式细胞,感染携带人野生型p５３,ＧＭ－ＣＳＦ和Ｂ７－１基因的重 组腺病毒后,观察这两种转基因癌细胞的生物学行为（包括药物敏感性０的变化,结果：药     

p53及B7重组痘苗病毒抗肿瘤的实验研究

目的：探索点突变 ｐ５３重组痘苗病毒诱导的抗瘤免疫反应及 Ｂ７对其加强作用,为重组抗原疫苗用于肿瘤免疫治疗提供实验依据。方法：选择人 １３５位 Ｃｙｓ→Ｔｙｒ点突变ｐ５３作为肿瘤相关抗原模型,观察以表达该点突变 ｐ５３的重组痘苗病毒ｒＶＶ－ｐ５３ＦＬ单独和联合表达 Ｂ７的重组痘苗病毒 ｒＶＶ－Ｂ７所诱导的 ＣＴＬ及对荷瘤小鼠的免疫保护和治疗作用。结果：以 ｒＶＶ－ｐ５３ＦＬ经静脉免疫 ＢＡＬＢ／ｃ小鼠能够诱导以 ＣＤＳ＋ Ｔ细胞为主的特异性 ＣＴＬ。ｒＶＶ－ｐ５３ＦＬ能够保护部分小鼠免遭肿瘤细胞的攻击。以ｒＶＶ－ｐ５３ＦＬ治疗荷瘤小鼠,可显著延长小鼠平均存活期。ｒＶＶ－Ｂ７与ｒＶＶ－ｐ５３ＦＬ以１：１的比例混合接种可部分加强ｒｖｖ－ｐ５３ＦＬ的治疗作用。结论：肿瘤细胞内过度表达的突变Ｐ５３蛋白可作为ＣＴＬ识别和攻击的靶抗原,以突变Ｐ５３蛋白作为肿瘤抗原的疫苗可诱导机体产生 ｐ５３特异性的抗瘤免疫反应。共刺激分子 Ｂ７可加强肿瘤抗原诱导的抗瘤免疫反应。     

重组人GM—CSF逆转录病毒基因转移系统的建立及其在人肿瘤细胞中的稳定…

建立逆转录病毒介导的ＧＭ－ＣＳＦ基因转移系统,为ＧＭ－ＣＳＦ基因悠我肿瘤细胞疫苗研究奠定实验基础。应用基因重组技术将ＧＭ－ＣＳＦｃＤＮＡ克隆于逆转录病毒载体ｐＤＯＲｎｅｏ,获得重组载体ｐＤＯＲＧＭ。经脂质体介针其转染于病毒包装细胞系ＰＡ３１７细胞,经ＮＩＨ３Ｔ３细胞检测病毒滴度,ＮＩＨ３Ｔ３放大实验检测辅助病毒,并用同熵度病毒感染人乳腺癌细胞系ＭＣＦ－７。     

p53抗原肽及B7重组痘苗病毒抗肿瘤的研究

目的：探索点突变ｐ５３抗原肽重组痘苗病毒诱导的抗瘤免疫效应,以及Ｂ７对之的加强作用,为重组抗原肽疫苗用于肿瘤免疫治疗提供实验依据。方法：选择人１３５位Ｃｙｓ→Ｔｙｒ点突变ｐ５３为肿瘤相关抗原模式,观察包含该点突变ｐ５３抗原肽ｐ５３１２５～１４５的重组痘苗病毒ｒＶＶ－ｐ５３Ｍ单独和联合应用表达Ｂ７的重组痘苗病毒ｒ－ＶＶ－Ｂ７诱导的ＣＴＬ及对荷瘤小鼠的免疫保护和治疗效应。结果：以ｒＶＶ－ｐ５３Ｍ经静脉免疫ＢＡＬＢ／ｃ小鼠能够诱导以ＣＤ８＾＋Ｔ细胞为主的特异性ＣＴＬ。ｒＶＶ－ｐ５３Ｍ能以１：１的比例混合接种可加强ｒＶＶ－ｐ５３Ｍ诱导的抗瘤免疫反应。结论：以痘苗病毒为载体的ｐ５３抗原肽疫苗代夫人工合成抗原肽,有潜在临床应用价值,共刺激分子Ｂ７与肿瘤抗原相结合是一种有效的免疫治疗策略。     

双苄基异喹啉生物碱蝙蝠葛碱与蝙蝠葛苏林碱逆转多药耐药性的比较研究

比较了种双苄基异喹啉生物碱蝙蝠葛碱，蝙蝠葛苏林碱及维拉帕米逆转多药耐药性的作用结果，ＤＲＣ，ＤＲＳ和ＶＲＰ在获得性耐药的ＭＣＦ０７／Ａｄｒ和ＫＢＶ２００细胞，以且生耐药的ＢＥＬ－７４０２细胞对ＡＤＲ和ＶＣＲ均有明显增敏作用，且作用呈剂量依赖性。     

外源野生型p53基因表达对人肺癌细胞药物敏感性影响

了解外源野生型ｐ５３（ｗｉｌｄ－ｔｙｐｅ ｐ５３,ｗｔｐ５３）基因表达对人大细胞肺癌８０１－Ｄ细胞对化疗药物敏感性的影响。方法选用Ｐ５３基因突为的人大细胞肺癌８０１－Ｄ系。用脂质体介导构建的含ｗｔｐ５３基因载体转染８０１－Ｄ细胞,ＰＣＲ检测外源ｗｔｐ５３在宿主细胞存在情况,＾３Ｈ－ＴｄＲ掺入法测定８０１－Ｄ细胞转染ｗｔｐ５３后药物敏感性变化,流式细胞仪（ＦＡＣＳ）分析细胞死亡情况。结果转染ｗｔｐ５     

重组人肿瘤坏死因子抗卵巢癌的体内外实验研究

以ＭＴＴ法测试发现重组人肿瘤坏死因子（ｒＨＴＮＦ－α）对体外培养的人卵巢癌细胞系ＯＶＣＡＲ３和ＣＡＯＶ３有较强的细胞毒效应。同时还测试了五种化疗药物５－Ｆｕ、ＣＴＸ、ＶＣＲ、ＤＤＰ、ＫＳＭ对这两种细胞的细胞毒性。在化疗药物浓度参照临床用药剂量制定的条件下，五种化疗药物对这两种细胞的细胞毒性均明显低于ｒＨＴＮＦ－α。因此，ｒＨＴＮＦ－α抗卵巢癌的潜力值得重视。形态学研究表明，ｒＨＴＮＦ－α首先引起卵巢癌细胞的线粒体和内质网的破坏，继之才出现细胞膜损伤和细胞裂解。实验还证实，ｒＨＴＮＦ－α与ＤＤＰ或ＫＳＭ联合均有一定的协同抗癌效应。在体外实验的基础上，作者对人卵巢癌细胞ＯＶＣＡＲ３的裸鼠移植瘤模型进行了实验性治疗。结果显示，ｒＨＴＮＦ－α单独应用有显著的抗移植瘤作用，与ＫＳＭ合用有较强的协同效应。     

腺病毒介导的p53基因对喉癌细胞生长的抑制作用

目的探索ｐ５３基因在喉癌基因治疗方面的可行性。方法以人喉癌细胞系Ｈｅｐ－２为实验对象,将载有人野生型ｐ５３ｃＤＮＡ并含巨细胞病毒（ＣＭＶ）启动子的重组腺病毒（Ａｄ５ＣＭＶ－ｐ５３）感染Ｈｅｐ－２细胞及肿瘤组织,体内外实验观察Ａｄ５ＣＭＶ－ｐ５３对Ｈｅｐ－２细胞生长的影响。结果当Ａｄ５ＣＭＶ－ｐ５３在１００ＭＯＩ效靶比时,全部Ｈｅｐ－２细胞得到转染。感染２天后ｐ５３蛋白表达达到高峰,Ｈｅｐ－２生长受到明显的抑制。Ａｄ５ＣＭＶ－ｐ５３感染Ｈｅｐ－２细胞在裸鼠中失去致瘤性。瘤内注射Ａｄ５ＣＭＶ－ｐ５３后,荷瘤裸鼠的肿瘤体积明显减小。结论Ａｄ５ＣＭＶ－ｐ５３转导野生型ｐ５３基因可能是一种有效的喉癌基因治疗途径。     

腺病毒介导野生型p53基因对人白血病细胞系HL-60、K_(562)生长的抑制作用

目的探索肿瘤抑制基因ｐ５３对人白血病细胞系的生长抑制作用和促凋亡作用。方法选用含野生型ｐ５３基因的重组腺病毒载体,体外感染人白血病细胞系ＨＬ－６０、Ｋ５６２。通过细胞生长曲线,ＤＮＡ检测及流式细胞仪分析检测转染细胞的增殖受抑制和细胞凋亡的发生。结果ＰＣＲ检测转染后ＨＬ－６０和Ｋ５６２细胞ｐ５３ｃＤＮＡ表达。转染后Ａｄ／ｐ５３病毒对ＨＬ－６０细胞和Ｋ５６２细胞的生长有抑制作用。随着Ａｄ／ｐ５３病毒浓度加大,ＨＬ－６０和Ｋ５６２细胞的ＯＤ值越低即生长抑制程度越大。经Ａｄ／ｐ５３病毒转染的ＨＬ－６０和Ｋ５６２细胞均有明显的细胞凋亡峰出现,对照细胞则无。细胞周期分析无明显异常发现。结论重组腺病毒载体介导的野生型ｐ５３基因在体外对人白血病细胞系ＨＬ－６０和Ｋ５６２的生长有抑制作用,能导致细胞凋亡的发生。     

野生型p53基因抑制膀胱癌细胞株HTB9生长及其机理的研究

目的：探讨野生型ｐ５３基因抑制膀胱癌生长的作用及其机理,方法：将野生型ｐ５３基是组腺病毒载体转染入膀胱癌细胞ＨＴＢ９,观察细胞生长曲线,细胞周期变化及ＤＮＡ合成情况,应用ＲＴ－ＰＣＲ检测ｐ２１ｍＲＮＡ水平,应用免疫组化法检测ｐ２１蛋白表达水平。结果：野生型ｐ５３基因导入可抑制ＨＴＢ９细胞生长和ＤＮＡ合成（ＡｄＣＭＶｐ５３）,流式细胞仪显示,ＤＮＡ合成前期或静止静的细胞比例增高,而合成期细胞比例下降,另外,ＡｄＣＭＶｐ５３还提高了ｐ２１的ｍＲＮＡ和蛋白水平,结论：腺病毒载体介导的野生型ｐ５３基因转梁对于膀胱可能是很有前途的基因治疗方法。     

Effect on biological behavior of chemotherapy-resistant tumor cells by human wild-type p53, GM-CSF and B7-1 genes via recombinant adenovirus

　Objective To explore the effect on biological behavior of chemotherapy-resistant tumor cells by human wild-type p53, GM-CSF and B7-1 genes mediated via recombinant adenovirus. Methods p53-abnormal KB-v200 (VCR resistant) and KB-s (VCR sensitive) cell lines were used as model tumor cells, which are resistant and sensitive to chemotherapeutic drugs respectively. After infected with recombinant adenovirus carrying human wild-type p53, GM-CSF and B7-1 genes, changes in biological behavior (including drug sensitivity) of these two kinds of gene-transduced cancer cells were observed. Results Both of the cell lines were susceptible to adenovirus, all of three exogenous genes (p53, GM-CSF and B7-1) could be effectively expressed in these cell lines, their growth was suppressed, and apoptosis was induced. The drug-pumping-out function of Pgp glycoprotein on the cytomembrane of drug-resistant KB-v200 cells was markedly affected 48h after transfection of the recombinant adenovirus, revealed by increase of the amount of rhodamine 123 accumulation in the cells. The MTT assay also indicated the reversal of their sensitivity to VCR drugs. In vivo experiment in nude mice it was demonstrated reduction of tumorigenicity of the KB-v200 cells or KB-s cells infected with the recombinant adenovirus, and increase of their sensitivity to VCR. Conclusion The clinical application of this recombinant adenovirus carrying agents might be more effective in treatment of tumors with multidrug resistance (MDR).     

腺病毒介导多基因在肺癌细胞中的表达及致凋亡效应

目的:观察重组腺病毒介导入野生型p53,B7-1和GM-CSF基因在肺癌细胞中的表达及致调亡效应.方法:以复制缺陷型重组腺病毒为载体,将入野生型P53,B7-1和GM-CSF基因导入肺癌细胞,分别以免疫组织化学法、流式细胞分析法和ELJSA法检测了外源基因在肺癌细胞中的表达,通过苔盼蓝染色后计数活细胞数绘制细胞生长曲线,末端标记法检出细胞凋亡.结果:观察到重组腺病毒分导的多种外源基因均可在肺癌细胞中高效表达,并可明显抑制肺癌细胞增殖并诱导其凋亡.结论:以上工作为进一步开展肺癌的多基因治疗打下了基础.     

Growth suppression and immunogenicity enhancement of Hep-2 or primary laryngeal cancer cells by adenovirus-mediated co-transfer of human wild-type p53, granulocyte-macrophage colony-stimulating factor and B7-1 genes

Co-transfer of immunomodulatory and antiproliferative genes may be the basis for new strategies to potentiate tumor regression. In this study, we evaluated the in vitro effect of the introduction of human wild-type p53, granulocyte-macrophage colony-stimulating factor (GM-CSF), and B7-1 genes via recombinant adenovirus on the growth and immunogenicity of Hep-2 or primary laryngeal cancer cells. By the introduction of wild-type p53 gene, the growth of Hep-2 cells was inhibited via enhanced apoptosis. By the introduction of GM-CSF and B7-1 genes, the immunogenicity of cancer cells was enhanced. Significant proliferation of tumor infiltrating lymphocytes (TILs) and tumor-specific cytotoxicity of cytotoxic T lymphocytes (CTLs) were induced in vitro. Furthermore, the combinative effect of GM-CSF and B7-1 was even more evident than that of any one of them singly. These results suggest that the co-transfer of human wild-type p53, GM-CSF and B7-1 genes into tumor cells via recombinant adenovirus may be further developed into a potential combination gene therapy strategy for cancer.     

Gene therapy for human nasopharyngeal carcinoma by adenovirus-mediated transfer of human p53, GM-CSF, and B7-1 genes in a mouse xenograft tumor model

Incidence of nasopharyngeal carcinoma (NPC) remains high in endemic regions. Prevention of tumor recurrences and metastases is a crucial approach to improve therapeutic outcome in NPC patients. In this study, we investigated the effects of the cotransfer of the tumor suppressor gene, p53, in combination with the immunostimulatory genes, GM-CSF and B7-1, on tumor regression and subsequent tumor recurrence. We constructed a recombinant adenovirus carrying human wild-type p53, granulocyte-macrophage colony-stimulating factor (GM-CSF), and B7-1 genes (Ad-p53/GM-CSF/B7-1), which mediated high-level expression of these three genes in NPC CNE-1 cells. Ad-p53/GM-CSF/B7-1 infection inhibited the growth of CNE-1 cells and induced tumor-specific cytotoxic T-lymphocytes (CTLs) in vitro. In CNE-1 xenograft tumor models in huPBL-nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, an intratumoral injection of Ad-p53/GM-CSF/B7-1 resulted in a reduced tumor burden, compared to normal saline (NS) and Ad-p53 controls. Tumors in the Ad-p53/GM-CSF/B7-1 group displayed diffuse necrosis and infiltration of human T-cells. Further, the tumor occurrence of CNE-1 cell rechallenge largely decreased after the primary tumor was intratumorally injected with Ad-p53/GM-CSF/B7-1 in the HuPBL-NOD/SCID mice model. Only 2 of 8 (25%) animals in the Ad-p53/GM-CSF/B7-1 group had developed measurable tumors, which demonstrated extensive necrosis and much more human T-cell infiltration, compared to 5 of 7 (71%) in the NS and Ad-p53 groups. Therefore, the adenovirus-mediated introduction of p53, GM-CSF, and B7-1 genes could improve local control and prevent the recurrence or metastases of NPC tumors, which suggests a potential therapeutic value in NPC treatment.     

Synergistic effects of adenovirus expressing wild-type p53 on chemosensitivity of non-small cell lung cancer cells

The infection of recombinant adenovirus expressing wild-type p53 (Ad-p53) to lung cancer cells that harbor mutant p53 genes improves their response to cis-diamminedichloroplatinum(II). In this study, we tested whether this improvement in response is also seen in wild-type p53 (wt-p53)-containing cancer cells and whether this phenomenon is universal with other commonly used chemotherapeutic agents, including etoposide, 7-ethyl-10-hydrocycamptothecin, paclitaxel, and docetaxel. Using a panel of 7 non-small cell lung cancer cell lines with wild-type (2) or abnormal (2 null, 3 point-mutated) p53, we examined in vitro cytotoxicity using a tetrazolium-based colorimetric assay (3-(4,5-diethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide assay) and analyzed the combined effects of Ad-p53 and chemotherapeutic agents using the isobologram method. Ad-p53 and DNA-damaging agents (cis-diamminedichloroplatinum(II), etoposide, and 7-ethyl-10-hydrocycamptothecin) showed synergistic effects in six of seven cell lines but additive effects against a p53-mutated cell line. In contrast, Ad-p53 showed additive effects with the antitubulin agents (paclitaxel and docetaxel) in all four of the cell lines tested. Furthermore, we examined this synergistic interaction between Ad-p53 and DNA-damaging agents by flow cytometric analysis and DNA fragmentation analysis. Both analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by DNA-damaging agents in six of seven cell lines. Our results suggest that Ad-p53 may synergistically enhance the chemosensitivity of the majority of non-small cell lung cancers to DNA-damaging agents due to augmentation of apoptosis.     

Adenoviral p53 gene therapy in head and neck squamous cell carcinoma cell lines

We reconstructed the recombinant p53-expressing adenovirus and examined its infections and effects in head and neck squamous cell carcinoma cell lines. Eight human head and neck squamous cell carcinoma cell lines were infected by the recombinant adenovirus harboring the lacZ gene (AxCAiLacZ) or the wild-type p53 gene (AxCAip53), and the effects were investigated. The eight cell lines were successfully infected by AxCAiLacZ at a level of more than 50%. The survival of all 8 squamous cell lines were inhibited in the range from 8 to 26.7% by only one treatment of the AxCAip53 infection. This result suggested that p53 gene therapy might become a useful tool in head and neck squamous cell carcinoma treatment.     

Generation of multidrug resistant lymphoma cell lines stably expressing P-glycoprotein

The objective of this study was to generate new P-glycoprotein (P-gp)-expressing multidrug resistant (MDR) cell lines by drug selection. Since our previous studies have been carried out with cells infected with a P-gp-containing vector, it was important to confirm our findings in cells generated by drug selection. In this report, we describe three B-lymphoma cell lines which became drug-resistant by stepwise exposure to vincristine (VCR): Raji cells resistant to 18 nM VCR (R18V), Namalwa cells resistant to 21 nM VCR (N21V) and DHL-4 cells resistant to 12 nM VCR (DHL-4/12V). Cells overexpressed P-gp and continued to express CD19, CD20 and CD22, all of which are targets for monoclonal antibody (MAb) therapy. The P-gp pump in these new cells was functional as determined by the efflux of Rhodamine 123 and DIOC2, and the three cell lines were resistant to several chemotherapeutic drugs. We further determined that their P-gp phenotype was stable in xenografted SCID mice and that the tumors were also resistant to chemotherapy. We will now use these new MDR cells to determine whether monoclonal antibodies against CD19 and -20 can reverse P-gp, as we previously demonstrated using Namalwa cells infected with a human mdr1 gene-containing retrovirus.     

Adenovirus-mediated Ink4a/ARF gene transfer significantly suppressed the growth of pancreatic carcinoma cells

OBJECTIVES: To determine the effects of adenovirus-mediated transfer of p14(ARF) and p16(INK4a) on growth and apoptosis of human pancreatic carcinoma cell lines. RESULTs: Pancreatic carcinoma cell lines, PC-7, PANC-1 and MIA PaCa-2 (p14(ARF)-/- and p16(INK4a)-/-), were used. PC-7 (p53 wt) and MIA PaCa-2 (p53 mt) cells infected with recombinant adenovirus vector expressing p14(ARF) gene (Adp14) showed significant inhibition of cell growth compared with control vector in vitro and in vivo, whereas PANC-1 cells (p53 mt) did not show such effects. Those three cell lines exhibited growth retardation and senescence phenotype after infected with the adenovirus vector containing p16(INK4a) gene (Adp16) in vivo and in vitro. CONCLUSIONS: Adenovirus-mediated transfer of p14(ARF) and p16(INK4a) produces significant growth suppression of pancreatic carcinoma cells in vitro and in vivo. The effects of Adp14 partly depend on the status of p53 gene in those cells.     

Late resistance to adenoviral p53-mediated apoptosis caused by decreased expression of Coxsackie-adenovirus receptors in human lung cancer cells

Adenovirus-mediated wild-type p53 gene transfer induces apoptosis in a variety of human cancer cells. Although clinical trials have demonstrated that a replication-deficient recombinant adenovirus expressing the wild-type p53 gene (Ad-p53) is effective in suppressing growth of non-small cell lung cancer (NSCLC), we often experienced late resistance to this treatment. To elucidate the mechanism of late resistance to Ad-p53 in human lung cancer cells, we generated 5 different resistant variants from p53-susceptible H1299 NSCLC cells by repeated infections with Ad-p53. We first examined the transduction efficiency of adenoviral vector by Ad-LacZ transduction followed by X-gal staining in parental and 5 resistant H1299 cell lines. Their sensitivity to viral infection decreased in correlation with the magnitude of resistance, and Ad-p53-mediated tumor suppression could be restored by dose escalation of Ad-p53 in the resistant variants. The expression of Coxsackie and adenovirus receptor (CAR) and alphaV integrins, which are cellular receptors for attachment and internalization of the virus, respectively, was next investigated in these cell lines. Flow cytometry revealed that alphaVbeta3 and alphaVbeta5 integrin expression was consistent, while p53-resistant cell lines showed that diminished CAR expression correlated with the magnitude of the resistance. Our results demonstrated that decreased CAR expression could be one of the mechanisms of late resistance to Ad-p53, which may have a significant impact on the outcome of adenovirus-based cancer gene therapy.