p53重组腺病毒联合顺铂或三氧化二砷对人膀胱癌EJ细胞的作用

目的：探讨p53重组腺病毒（Ad-p53）与顺铂（CDDP）或三氧化二砷（As2O3）联合应用提高膀胱癌疗效的可能性及其机制。方法：将Ad-p53[100感染强度（MOI）]与CDDP（0．5mg/L)或As2O3(2.0μmol/L）联合应用，通过细胞生长抑制实验、克隆形成实验、细胞周期分析、免疫组织化学分析以及裸鼠皮下移植瘤模型，观察其对膀胱癌EJ细胞的作用及机制。结果：与单独应用相比，Ad-p53与低剂量的CDDP或As2O3联合可明显抑制EJ细胞体外生长，诱导EJ细胞凋亡，EJ细胞G2／M期阻滞更明显；裸鼠体内肿瘤发生时间延迟，4周后肿瘤体积与单独应用时相比，差异有显著性（P＜0．05）。结论：基因治疗与化疗联合应用，可进一步提高膀胱癌的疗效。     

p53重组腺病毒联合增殖细胞核抗原反义寡核苷酸治疗膀胱癌的研究

目的：探讨重组腺病毒p53(Ad-p53)与增殖细胞核抗原反义寡核苷酸（PCNA-ASO）联合应用对膀胱癌的抑瘤效应。方法：用腺病毒将p53{感染强度（MOI）100}导入细胞，PCNA-ASO(1.6μmol/L)在脂质体（Lipofectin）介导下转染细胞，通过噻唑蓝比色法（MTT）、流式细胞术、克隆形成、建立移植瘤模型，探讨其体内外抗瘤活性。结果：Ad-p53与PCNA-ASO联合应用明显抑膀胱癌胞EJ(89.3%)和BIU-87(78.6%)生长，细胞克隆形成能力分别下降74.8％和67.5％，S期比率（EJ细胞11.4％，BIU-87细胞14.6％）明显降低，细胞生长受阻于G1期(EJ细胞62.2％，BIU-87细胞56.8％)；联合应用7d后，肿瘤体积较初始分别下降47.6％和36.4％。结论：Ad-p53联合PCNA-ASO可抑制膀胱癌细胞体内外生长，产生协同效应。     

p16重组腺病毒联合顺铂或三氧化二砷对人膀胱癌EJ细胞作用的实验研究

目的　探讨 p16重组腺病毒 (Ad p16 )与顺铂 (CDDP)或三氧化二砷 (As2 O3 )联合应用对人膀胱癌EJ细胞体内外生长的抑制效应及机制。　方法　将Ad p16与CDDP或As2 O3 联合应用 ,通过细胞生长抑制实验 ,克隆形成实验 ,细胞周期分析 ,免疫组织化学分析 ,以及裸鼠皮下移植瘤模型 ,观察其对人膀胱癌EJ细胞的作用及机制。　结果　与单独应用相比 ,Ad p16与低剂量的CDDP或As2 O3 联合应用可明显抑制EJ细胞的体外生长 ,诱导EJ细胞凋亡 ,明显阻滞EJ细胞G1期 ;裸鼠体内肿瘤发生时间延迟 ,4周后肿瘤体积与单独应用时相比差别有显著性意义 (P <0 .0 5 )。　结论　Ad p16与CDDP或As2 O3 联合应用 ,有可能进一步提高膀胱癌的治疗效果     

反义增殖细胞核抗原联合p16基因转染抑制膀胱癌细胞生长的研究

目的 探讨膀胱癌联合基因治疗的新策略。方法 反义增殖细胞核抗原(PCNA)联合p16转染膀胱癌细胞，观测共转染1—7d后癌细胞增殖活性、DNA合成速率、细胞周期时相、克隆形成能力、细胞凋亡和PCNA、p16基因表达情况。结果 联合转染后癌细胞PCNA表达减弱，p16表达显著增强，增殖活性抑制15．45％—68．47％(P＜0．01)，DNA合成速率减慢65．77％(P＜0．01)，细胞周期阻滞于G0／G1期，克隆形成率抑制64．49％(P＜0．01)，凋亡率为22．00％(P＜0．01)。结论 反义PCNA与p16共转染具有抑制增殖、诱导凋亡的双重作用，有望成为膀胱癌联合基因治疗的新途径。     

重组腺相关病毒介导抑癌基因联合诱导人肝癌细胞系凋亡与生长抑制研究

目的 观察腺相关病毒介导的抑癌基因p53,p16和p21联合治疗肝癌的可能性和效果。方法 用携带人野生型p53,p16和p21的腺相关病毒分别或联合转导人肝癌细胞系HLE，HepG2,QGY-7701,QGY-7703,BEL-7402,SMMC-7721，通过体外及裸鼠体内实验研究转基因的表达及对癌的促凋亡和生长抑制作用。结果 腺相关病毒介导的p53,p16及p21对人肝细胞肝癌细胞系均有明显的促凋亡作用，体外凋亡率约30％左右，体内生长抑制率30％－44％；联合感染肝癌细胞效果更明显，体外凋亡率达56％，体内癌生长抑制率65％。结论 腺相关病毒介导的外源抑癌基因p53，p16和p21不仅对多种肝癌细胞系均有诱导凋亡和抑制生长作用，联合应用治疗肝癌更具有明显的相互协同作用。进一步提高病毒滴度和纯度是腺相关病毒作为载体进行基因治疗的目标。     

野生型p53基因对不同内源性p53状态的肝癌细胞株生长的影响

目的探讨外源性野生型p53(wild-type p53,WT-p53)基因对具有不同内源性p53功能状态肝癌细胞生长的影响.方法通过表达WT-p53的重组腺病毒载体(Ad-p53),将p53基因导入具有不同内源性p53功能状态的肝癌细胞株Bel-7402、QGY-7701和PLC/PRF/5中,用MTT法检测Ad-p53对各细胞株生长的影响,流式细胞仪分析各组细胞中DNA含量及凋亡的比例.结果当取40、200、800 MOI值的表达p53的腺病毒分别转染PLC/PRF/5、Bel-7402和QGY-7701细胞时,72 h后行MTT检测,OD值分别为在 PLC/PRF/5细胞,空载体(Ad-LacZ)组0.98±0.16,Ad-p53组0.02±0.00(P＜0.01);在Bel-7402细胞Ad-LacZ组1.04±0.23,Ad-p53组0.10±0.02(P＜0.01);而在QGY-7701细胞Ad-LacZ组1.88±0.24,Ad-p53组1.86±0.19(P>0.05).细胞周期分析显示,在PLC/PRF/5,表现为既诱导细胞凋亡,又诱导细胞周期阻滞.在Bel-7402,表现为诱导细胞周期阻滞.结论外源性p53对肝癌细胞生长的抑制可能与内源性p53功能状态无关.     

GADD45β基因表达诱导对不同p53状态的肝癌细胞作用

目的：体外合成GADD45β基因全序列表达质粒后，研究GADD45β基因表达诱导对呈不同p53状态的肝癌细胞的作用及可能机制。方法：采用RT-PCR法获得GADD45β基因全序列，插入pDrive穿梭克隆载体和pIRES2-EGFP荧光表达载体后大量扩增获得DNA，结合p53全基因表达质粒pp53-EGFP转染HepG2、Hep3B细胞后，以[^3H]胸腺嘧啶脱氧核苷掺入法(^3H—Tdr)和细胞克隆形成法分析DNA合成变化及细胞生长能力；以双抗体夹心ELISA法测定TGF-β1表达变化。结果：成功合成GADD45β基因全序列和表达质粒，通过流式细胞仪收集转染阳性的荧光表达细胞能显著提高转染效率；转染GADD45β后．具有野生型p53基因的HepG2细胞的细胞克隆形成能力和DNA合成能力明显受到抑制，细胞凋亡明显增加，TGF-β1的表达亦明显受抑。与之相反，缺失p53基因的Hep3B需要同时共转染p53基因后，方出现抑制效应。结论：GADD45β基因能够有效抑制肝癌细胞的生长．其功能需要完整p53基因的辅助和(或)调控。GADD45表达和（或)功能异常，导致p53介导的DNA损伤修复途径异常或阻断，是肝脏细胞恶性转化及形成肿瘤的可能机制。     

重组分泌型内皮抑素腺相关病毒体内治疗膀胱癌

目的观察重组分泌型内皮抑素腺相关病毒（rAAV—ES）在裸鼠模型体内的抗肿瘤作用。方法以rAAV-ES转染（转染复数MOI=1×10^5）膀胱癌细胞（EJ）后建立裸鼠肿瘤模型，检测EJ细胞被转染后的成瘤率及肿瘤生长情况；裸鼠肌注rAAV—ES后检测内皮抑素在体内的表达；建立裸鼠肿瘤模型，检测全身应用rAAV-ES后抑制肿瘤发展的作用、对肿瘤微血管密度（MVD）的影响及其毒副作用。结果被rAAV-ES转染的EJ细胞成瘤率仅为对照组的2／5；体内实验证实肌注rAAV-ES后血清中内皮抑素长期高效表达（30～40）μg/L；全身应用后肿瘤生长速度减慢（32±9）d，瘤体微血管密度变低（8．30±3．14）／0．739mm^2，心脑组织学检查未见缺血和其他异常改变。结论rAAV-ES无毒副作用，可有效地抑制肿瘤的血管生成，从而抑制膀胱癌的发生、发展，其成功包装为原位基因治疗膀胱癌奠定了基础。     

血管内皮生长因子可溶性受体flt—1n3对裸鼠人膀胱癌生长的影响

目的；研究血管内皮生长因子（VEGF）受体flt-1脑外区基因抗肿瘤新生血管生成的作用。方法：采用脂质体转染技术将含有flt-1胞外区前三个IgG样结构区域的真核表达质粒pcDNA3.1/KDPn7转入人膀胱癌EJ细胞，用G418筛选得到稳定表达目的蛋白的细胞克隆，经固相结合实验筛先得到表达与VEGF特异结合的目的蛋白的细胞克隆，阳性细胞克隆进行PCR和RT－PCR鉴定。对裸鼠人膀胱癌组织切片进行免疫组化染色，定量分析微血管密度。结果：PCR和RT－PCR结果显示重组质粒转入EJ细胞及在转录水平表达；免疫组化的微血管密度实验组明显低于阴性对照组。结论：阻断VEGF／flt-1信号传导途径能够抑制肿瘤新生血管的形成，延缓肿瘤的生长速度。     

端粒酶启动子调控p53基因表达对T24细胞的影响

目的 探讨端粒酶催化亚基（hTERT）启动子调控p53基因在膀胱肿瘤细胞T24中表达对细胞的影响。方法 将成功构建的phTERT-p53载体，瞬时转染膀胱肿瘤T24细胞；应用流式细胞仪观察细胞凋亡率变化，透射电镜下观察细胞凋亡形态学改变。结果 hTERT启动子调控p53基因表达使细胞凋亡率明显升高[24、48h凋亡率：实验组15．42％、36．57％；转染绿色荧光蛋白基因（GFP）组5．16％、8．19％；阴性对照组4．49％、7．18％]，电镜下观察到典型凋亡改变。凋亡指数达25％。结论 hTERT启动子能够激活phTERT-p53载体表达p53，使肿瘤细胞凋亡增加。     

Preclinical study of adenoviral p53 gene therapy for esophageal cancer

An alteration of the p53 gene function is a major factor in the development of esophageal cancer. Recently, p53 gene therapy has been applied for clinical studies in lung cancer and head and neck cancer. However, no preclinical studies have yet demonstrated an anticancer effect of adenoviral-mediated wild-type p53 gene therapy on esophageal cancer. We herein evaluated the effect of p53 adenoviral gene therapy on human esophageal squamous cell carcinoma to test the ability of clinical application. A normal esophageal epithelial cell line (EN53F) and two human esophageal cancer cell lines (ECGI-10 and T.Tn) with a p53 alteration were used. The transduction efficiency, p53 protein expression, p21 protein expression, the induction of apoptosis, and growth suppression were assessed by using the recombinant adenoviral vector Ad5CMV-p53. The transduction efficiency was 60%–80% at 100 plaque-forming units (PFU)/cell and 80%–100% at 300 PFU/cell. A significant growth suppression following an Ad5CMV-p53 infection was observed in both cancer cell lines. A Western blot analysis confirmed the presence of both exogenous p53 protein expression and p21 protein induction. Apoptotic cell death was observed with TUNEL staining. T.Tn xenografts in nude mice transduced with Ad5CMV-p53 demonstrated significant growth suppression. These data suggest that Ad5CMV-p53 may thus be a potentially effective therapeutic agent for locally advanced esophageal cancer. Received: July 19, 2000 / Accepted: January 9, 2001     

外源性p16基因导入对人膀胱癌细胞的作用

目的 通过外源性野生型Ｐ１６基因,纯翕生缺失及具有内源性Ｐ１６基因表达的膀胱癌细胞系２５３Ｊ和ＥＪ,地肿瘤细胞的生长抑制作用,并探讨共作用机制。方法 构建Ｐ１６逆转逆转录病毒表达载体,经细胞ＰＡ３４１７包装,膀胱癌细胞系;应用Ｎｏｒｔｈｅｍ杂交及免疫细胞化学方法检测外源１６基因的表达;流式我仪检测细胞周期;原位细胞凋亡检测及透射电镜观察细胞凋亡;并对导入的Ｐ１６基因的细胞进行鼠致瘤能力及病理不特性     

Adenoviral p53 gene transfer in human bladder cancer cell lines: cytotoxicity and synergy with cisplatin

Mutations of the tumor suppressor gene p53 are common in bladder cancer. To determine whether p53 gene transfer would lead to decreased viability of bladder cancer cells, we studied the effect of p53 gene transfer in human bladder cancer cell lines with either mutant or wild-type p53. Bladder cancer cell lines 5637 and J82 (which express only mutant p53) and 253J-BV (which expresses wild-type p53) were transduced with vectors containing the β-galactosidase gene (Ad5-lacZ), wild-type human p53 gene (Ad5CMV-p53), or no foreign gene (DL312 or Ad5-polyA). X-gal staining of cells exposed to Ad5-lacZ showed that the adenoviral vector was capable of transducing each of the cell lines. Increases in p53, p21^waf1/cip1 and bax protein were demonstrated following exposure to Ad5CMV-p53, and there was a dose-dependent increase in the number of apoptotic cells. Cell viability was decreased in all three cell lines, although J82 was less sensitive than either 5637 or 253J-BV. To determine whether cisplatin increases sensitivity of J82 cells to Ad5CMV-p53, we performed median effect analysis for cisplatin combined with Ad5CMV-p53 or DL312. The combination index for cisplatin plus Ad5CMV-p53 revealed synergy, whereas cisplatin and DL312 were only additive. These results suggest that forced p53 gene expression is cytotoxic to human bladder cancer cells with either p53 mutant or wild-type background, and that combination with cisplatin is a potential method for overcoming resistance.     

FUNCTIONAL p53 MUTATION AS A MOLECULAR DETERMINANT OF PACLITAXEL AND GEMCITABINE SUSCEPTIBILITY IN HUMAN BLADDER CANCER

PURPOSE: Paclitaxel and gemcitabine are promising new agents for treatment of human bladder cancer. We determine how the presence or absence of p53 function impacts the cytotoxic effects of these chemotherapeutic agents in human bladder cancer. MATERIALS AND METHODS: The J82 human bladder cancer (TCC) cell line was transfected with a temperature sensitive p53 (tsp53) mutant that functions as mutated p53 at 37C but functions as wild-type (normal) p53 at 32C. Susceptibility of these inducible p53 TCC cells to paclitaxel and gemcitabine induced cytotoxicity was evaluated and kill significance determined between sub-lethal and lethal doses. RESULTS: Significant paclitaxel dose dependent cytotoxicity was observed in J82 TCC cells lacking normal p53 and tsp53 transfected cells at 37C, which was the mutant p53 temperature in transfectants between maximal and minimal kill concentrations for either (p <0.001). Likewise, significant cytotoxicity was observed in parental J82 TCC at 32C (p <0.001), while restoration of p53 function in tsp53 transfected cells on shift to 32C abrogated significant dose dependent cytotoxicity. Gemcitabine caused significant cell death in the cell lines incubated at either temperature and, thus, was equally effective regardless of cellular p53 function (p <0.001, respectively). CONCLUSIONS: Paclitaxel requires functionally mutated p53 to induce cell death in human bladder cells, indicating that it may be more effective against TCC with p53 mutations than against TCC, which lacks p53 abnormalities, while gemcitabine is effective regardless of p53 function. These findings provide a rationale for selecting chemotherapy based on the p53 status of individual bladder cancers.     

Differential involvement of the Fas receptor/ligand system in p53-dependent apoptosis in human prostate cancer cells

BACKGROUND: The objective of this study was to characterize the involvement of the Fas receptor/ligand system in p53-dependent apoptosis in human prostate cancer cells. METHODS: The effects of adenovirus-mediated p53 gene transfer (Ad5CMV-p53) into human prostate cancer LNCaP, DU145, and PC3 cells on their growth, apoptosis and Fas receptor/ligand expression were examined by the MTT assay, DNA fragmentation assay, and Northern blot analysis, respectively. The sensitivity of these cells to an agonistic anti-Fas receptor antibody (CH11) and the effects of an antagonistic anti-Fas ligand antibody (4H9) on Ad5CMV-p53-induced apoptosis were analyzed by the MTT assay and DNA fragmentation assay. RESULTS: Ad5CMV-p53 treatment resulted in substantial growth inhibition, induction of apoptosis and up-regulation of Fas receptor as well as Fas ligand mRNA expression in LNCaP, DU145 and PC3 cells. Despite the abundant expression of Fas receptor in all of these cells, CH11 induced apoptosis only in PC3 cells. Furthermore, 4H9 partially blocked the apoptosis induced by Ad5CMV-p53 in PC3 cells, but not in LNCaP and DU145 cells. CONCLUSIONS: The Fas receptor/ligand system is differentially involved in p53-dependent apoptosis in prostate cancer cells; therefore, reintroduction of wild-type p53 into prostate cancer cells may induce apoptosis through Fas receptor/ligand interaction as well as through an alternative pathway.     

Growth inhibitory effect of p21 and p53 containing adenoviruses on transitional cell carcinoma cell lines in vitro and in vivo

Altered p53 expression has been demonstrated in the majority of advanced transitional cell carcinoma (TCC) of the bladder tumors. The objective of this investigation was to examine the effect of the introduction of a p53 or p21^(WAF1/CIP1) adenovirus on the proliferation and apoptosis of various human TCC cell lines in vitro and in vivo. Proliferation was measured by ³H-thymidine incorporation. Apoptosis was measured by DNA fragmentation and bax expression. We also examined the effect of ex vivo introduction of the p21^(WAF1/CIP1) or the p53 gene on growth of the T24 TCC cells and UMUC-3 TCC cells introduced subcutaneously into athymic nude mice. We found that although the effect of the p21-adenovirus on the proliferation of various TCC lines varied with each individual cell line, there was a substantial growth inhibition observed (greater than 80% growth inhibition) in seven of the eight TCC cell lines at the highest viral dosage. In contrast, after 24 h, the highest dosage of the p53-adenovirus produced only a heterogeneous decrease in proliferation compared to the highest dose of the p21^(WAF1/CIP1)-adenovirus (40–90%). In ex vivo experiments, no tumors were found in nude mice injected subcutaneously with either TCC cell line exposed in vitro to the AdSCMV-p21^(WAF1/CIP1) or AdSCMV-p53 viruses before three weeks. There was a threefold decrease in tumor square area at week 5 in the Ad5CMV-p21^(WAF1/CIP1) or Ad5CMV-p53 TCC cells injected mice (p<0.001, p<0.009) compared to either mock or Ad5CMVLacZ TCC bladder tumor cells. These data suggest that significant portion of the effect of altered p53 on TCC phenotype may be mediated through the p21^(WAF1/CIP1) pathway. Thus, the restoration of p21^(WAF1/CIP1) function in this tumor system may be a beneficial therapeutic strategy.