重组人p53腺病毒注射液联合顺铂对卵巢癌细胞生长及凋亡的影响

目的:探讨重组人p53基因腺病毒(recombinant adenovirus-p53,rAd-p53)注射液联合顺铂(cisplatin, DDP)对卵巢癌细胞株生长及细胞凋亡的影响.方法:利用MTT、FCM和Western 印迹法比较单独采用rAd-p53、DDP及2者联合用药对卵巢癌细胞株SKOV-3和CAOV-3细胞生长抑制、细胞周期、细胞凋亡以及p53蛋白表达的影响.结果: rAd-p53注射液对卵巢癌细胞株的生长有抑制作用,并呈时间、剂量依赖性关系;联合用药对卵巢癌细胞株生长抑制作用更显著(P<0.01),联合用药时不同的用药顺序对卵巢癌细胞株生长抑制无明显差异(P>0.05).联合用药72 h后,卵巢癌细胞株细胞周期明显阻滞于G0/G1期,S期比例明显减少,且细胞凋亡率显著升高(P<0.01).rAd-p53作用于卵巢癌细胞株72 h后,有明显的p53蛋白表达.结论:rAd-p53能将外源性野生型p53基因导入卵巢癌细胞基因组,使之表达p53蛋白,从而使肿瘤细胞的细胞周期阻滞于G0/G1期,抑制细胞生长,并促进细胞凋亡.rAd-p53与DDP联合应用,其抗肿瘤效应较单药用药更加显著.     

Growth inhibition of human laryngeal cancer cell with the adenovirus-mediated p53 gene

Mostmalignanciesarediseasegeneratedwithaprocessofgeneticalteration.Basedonthistheory,newapproachestocancertherapyarebeingdevel0ped.Oneoftheseisgenetherapy.Amongthegeneshavingtherapeuticpotentialforcancertreatment,thep53tumorsuppressorgenehasbeenmostextensivelystudied.[l]Wild-typep53hasbeenshownt0beinvolvedintranscirptionalregulation.ItarrestscellsattheGllScheckpoint,blocksDNAreplicati0nandinducesap0ptosis."'Recentstudieshavesh0wnthatwild-typep53genecansuppressthegrowthofsomehumancancercelll…     

重组人p53腺病毒注射液(今又生)对人肺腺癌细胞生长及化疗敏感性研究

背景与目的在肺癌中,p53突变是最常见的基因改变之一,p53基因的突变常导致细胞对化疗不敏感。有研究表明导入野生型p53基因能增加化疗药物的敏感性。本研究的目的是探讨重组人p53腺病毒注射液(Adp53,今又生,Gendicine)对人肺腺癌细胞生长及化疗敏感性的影响。方法将重组腺病毒载体所携带的野生型p53基因导入人肺腺癌细胞株GLC82(含突变型p53基因)及A549(含野生型p53基因),并联合应用化疗药物顺铂(DDP),通过Westernblot法分析外源野生型p53基因在细胞内的表达,MTT法和流式细胞术观察对细胞生长及细胞周期、凋亡的影响。结果通过Westernblot证实了外源p53基因能在GLC82及A549细胞中高效表达。MTT法观察到Adp53对肺癌细胞的抑制作用呈时间依赖性和剂量依赖性效应。100MOIAdp53与0.5mg/LDDP联合应用后72h,对A549细胞的生长抑制率达43.13%±0.72%,显著高于单用Adp53组(23.44%±0.54%,P<0.001)和DDP组(14.17%±1.39%,P<0.001);对GLC82细胞生长的抑制率达63.73%±0.92%,显著高于单用Adp53组(41.51%±0.59%,P<0.001)和DDP组(56.11%±1.12%,P<0.001)。流式细胞术检测结果显示,Adp53与DDP联合应用能使细胞阻滞于G0G1期,S期细胞比例明显减少。Adp53+DDP组A549细胞凋亡率为28.99%±1.07%,显著高于单用Adp53组(15.35%±1.31%,P<0.001)和DDP组(1.74%±0.77%,P<0.001);Adp53+DDP组GLC82细胞凋亡率为62.98%±2.43%,显著高于单用Adp53组(20.88%±0.71%,P<0.001)和DDP组(6.91%±1.52%,P<0.001)。结论重组人p53腺病毒注射液能抑制肺腺癌细胞的生长,并不受内源性p53状态的影响。它与抗癌药DDP联用能显著增加肺腺癌细胞的化疗敏感性。     

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。     

Mitomycin C and cisplatin enhanced the antitumor activity of p53-expressing adenovirus in cervical cancer cells

This study investigated the enhanced antitumor activity of Ad5-p53 in combination with mitomycin C (MMC) or cisplatin (DDP) in cervical cancer cell lines SiHa and C-33A. MMC and DDP inhibited the growth of SiHa and C-33A cells in a dose-dependent manner, and the combination of MMC or DDP with Ad5-p53showed a stronger growth inhibition than those treated with either Ad5-p53, MMC, or DDP alone. As evidenced by the formation of the ∼200 bp DNA ladder and the appearance of sub-G1 peak, both MMC and DDP induced apoptosis in cervical cancer cells. Western blot analysis of p53 showed that MMC/DDP did not induce the increase of p53 protein in SiHa cells nor the increase of the cellular and nuclear p53 protein in Ad5-p53 transfected Saos-2 cells. Taken together, these results suggested that the combination of Ad5-p53and MMC/DDP may serve as an effective therapeutic regime for human cervical cancer treatment.     

外源性p53基因增加卵巢癌细胞对顺铂的敏感性

 目的 探讨外源性ｐ５３基因转染对人卵巢癌细胞株的化疗敏感性的影响。方法用脂质体介导的转染技术，将人野生型ｐ５３基因的真核表达载体导入不表达ｐ５３的卵巢癌ＳＫＯＶ－３细胞中，经Ｇ４１８筛选，Ｎｏｒｔｈｅｒｎｂｌｏｔ及Ｗｅｓｔｅｒｎｂｌｏｔ鉴定后，观察其对顺铂作用后的 ＳＫＯＶ－３细胞的集落形成及凋亡的影响。结果 外源性Ｐ５３基因在转染细胞中有效表达，并增强了顺铂对ＳＫＯＶ－３细胞集落形成的抑制作用及促进了顺铂诱导的细胞凋亡。结论 外源性ｐ５３基因能增加卵巢癌细胞对顺铂的敏感性，两者联合作用能更大程度地杀灭肿瘤细胞。     

p53调控的肺癌细胞顺铂敏感性相关基因片段的分离和克隆

目的 克隆野生型p53(wtp53)影响人大细胞肺癌801一D细胞系顺铂敏感性相关基因片段。方法 采用转基因方法补偿801-D细胞p53功能，分别提取经IC50浓度顺铂刺激的转染wtp53细胞、空载细胞和未经顺铂作用的转wtp53细胞RNA，用银染mRNA差异显示方法分离差异表达基因，反转录点杂交和巢式PCR进行确证。阳性片段进行克隆及序列分析。结果 获得6条阳性片段，2条含有开放阅读框架(ORF)，其中1条片段部分与核糖核苷还原酶α链有56％同源性，另一片段D1含有一个长ORF，与已知cDNA序列同源率很低。结论 p53对801-D细胞顺铂敏感性的影响有明显的基因水平改变，可能诱导了相关基因表达。     

逆转录病毒介导p53基因对食管癌细胞株的生长抑制作用

目的观察人类野生型p53(wt p53)基因对人食管癌细胞系的抑制作用.方法用逆转录病毒为载体将外源性wt p53基因导入人食管癌细胞系ECA109,通过体外及小鼠体内实验研究转入基因的表达及对肿瘤的抑制作用.结果 p53在转染细胞ECA109/p53中表达水平提高.外源性wt p53基因的导入和表达能使ECA109细胞的生长速率减低,软琼脂集落形成能力下降,G0 G1期细胞比例增加、S期细胞比例降低,凋亡指数升高,裸鼠体内成瘤能力明显下降.结论逆转录病毒载体介导的外源性野生型p53能够抑制人食管癌细胞生长.     

MLH1 expression sensitises ovarian cancer cells to cell death mediated by XIAP inhibition

Background:

The X-linked inhibitor of apoptosis protein (XIAP), an endogenous apoptosis suppressor, can determine the level of caspase accumulation and the resultant response to apoptosis-inducing agents such as cisplatin in epithelial ovarian cancer (EOC). In addition, the mismatch repair protein, hMLH1, has been linked to DNA damage-induced apoptosis by cisplatin by both p53-dependent and -independent mechanisms.

Methods:

In this study, hMLH1 expression was correlated with clinical response to platinum drugs and survival in advanced stage (III–IV) EOC patients. We then investigated whether MLH1 loss was a determinant in anti-apoptosis response to cisplatin mediated by XIAP in isogenic and established EOC cell lines with differential p53 status.

Results:

The percentage of cells undergoing cisplatin-induced cell killing was higher in MLH1-proficient cells than in MLH1-defective cells. In addition, the presence of wild-type hMLH1 or hMLH1 re-expression significantly increased sensitivity to 6-thioguanine, a MMR-dependent agent. Cell-death response to 6-thioguanine and cisplatin was associated with significant proteolysis of MLH1, with XIAP destabilisation and increased caspase-3 activity. The siRNA-mediated inhibition of XIAP increased MLH1 proteolysis and cell death in MLH1-proficient cells but not in MLH1-defective cells.

Conclusion:

These data suggest that XIAP inhibitors may prove to be an effective means of sensitising EOC to MLH1-dependent apoptosis.     

外源p53对人肺癌细胞生长的抑制

目的　观察外源性野生型p53对有p53基因突变的人肺癌细胞系生长的影响。方法　用PCR SSCP及DNA测序 ,选择p53突变的人肺巨细胞癌系 80 1 D。构建野生型p53表达质粒PZiP p53。用基因枪介导外源基因。经G41 8筛选得到转染细胞系 80 1 D p53。用PCR检测外源基因 ,观察转染细胞恶性生长的变化。结果　转染细胞系 80 1 D p53体外长期传代有外源性p53基因存在 ,转染细胞生长明显受到抑制 ,集落形成抑制率达 96% ,裸鼠异种移植致瘤性降低 ,肿瘤生长明显缓慢。结论　外源性野生型p53经基因枪导入有p53基因突变的人肺癌细胞后可长期存在于转染细胞中 ,且明显抑制所转染的癌细胞的恶性生长。     

p53-dependent radioresistance in ovarian carcinoma cell lines

In the present study, we investigated the radiosensitivity profiles of three established human ovarian carcinoma cell lines, PA-1, Caov-3, and SK-OV-3, using the adenosine triphosphate-cell viability assay (ATP-CVA). We have correlated radioresponsiveness with the p53 status and the p53 accumulation after irradiation as well as with the Bcl-2 expression and the growth rate of these cell lines. The p53 status was examined by immunocytochemistry and a functional assay (functional analysis of separated alleles in yeast, FASAY); the p53 accumulation was determined by immunocyotochemistry and flow cytometry. Futhermore, the Bcl-2 expression before and after irradiation was examined by immunocytochemistry. PA-1, expressing wild-type p53, showed an unequivocal accumulation of p53 protein following exposure to irradiation. This cell line was found to be strongly sensitive to irradiation. The two p53 mutant cell lines Caov-3 and SK-OV-3 showed radioresistance at different degrees and irradiation did not result in p53 accumulation. None of the cell lines examined expressed Bcl-2 protein and no change was seen after irradiation. Furthermore, the most sensitive cell line to irradiation, PA-1, showed the highest proliferative activity, while Caov-3 and SK-OV-3, the more resistant cell lines, exhibited lower growth rates. Our findings indicate that the presence of p53 protein is a possible determinant for the cytotoxicity induced by irradiation in the investigated ovarian carcinoma cell lines. Bcl-2 expression does not seem to determine the response to irradiation in these cell lines. Additionally, an association between radioresponsiveness and the growth rate is suggested in PA-1, Caov-3, and SK-OV-3.     

Akt promotes cisplatin resistance in human ovarian cancer cells through inhibition of p53 phosphorylation and nuclear function

Resistance to cisplatin-based chemotherapy is a major cause of treatment failure in human ovarian cancer. Wild-type TP53 status is often, but not always, associated with cisplatin sensitivity, suggesting that additional factors may be involved. Overexpression/activation of the phosphatidylinositol-3-kinase/Akt pathway is commonly observed in ovarian cancer, and Akt activation is a determinant of chemoresistance in ovarian cancer cells, an effect that may be due, in part, to its inhibitory actions on p53-dependent apoptosis. To that end, we examined the role and regulation of p53 in chemosensitive ovarian cancer cells, as well as in their chemoresistant counterparts, and investigated if and how Akt influences this pathway. Cisplatin induced apoptosis in chemosensitive, but not chemoresistant cells, and this was inhibited by downregulation of p53. Cisplatin upregulated PUMA in a p53-dependent manner, and the presence of PUMA was necessary, but not sufficient for cisplatin-induced apoptosis. p53 was phosphorylated on numerous N-terminal residues, including Ser15, Ser20, in response to cisplatin in chemosensitive, but not chemoresistant cells. Furthermore, activation of Akt inhibited the cisplatin-induced upregulation of PUMA, and suppressed cisplatin-induced p53 phosphorylation, while inhibition of Akt increased total and phospho-p53 contents and sensitized p53 wild-type, chemoresistant cells to cisplatin-induced apoptosis. Finally, mutation of Ser15 and/or Ser20, but not of Ser37, to alanine significantly attenuated the ability of p53 to facilitate CDDP-induced apoptosis, and this was independent of PUMA expression. These results support the hypothesis that p53 is a determinant of CDDP sensitivity, and suggest that Akt contributes to chemoresistance, in part, by attenuating p53-mediated PUMA upregulation and phosphorylation of p53, which are essential, but independent determinants of sensitivity to CDDP-induced apoptosis.     

Induction of p53 and drug resistance following treatment with cisplatin or paclitaxel in ovarian cancer cell lines

Treatment failures result from resistance to chemotherapy in ovarian cancer. The effect of cisplatin and paclitaxel treatments on chemosensitivity was studied in ovarian cancer cells developed from a patient with stage IIIC disease. Cells (UL-3A, UL-3B) that recovered from cisplatin (Cis) and paclitaxel (Tax) treatments showed higher levels of p53, mdr-1 and chemoresistance than untreated controls. EC50 values of Cis and Tax for UL-3A clones were 7.2-34.6, average 20.9 microg/ml, while UL-3B clones ranged from 11.8-252.0 microg/ml, with a mean value of 73.2 microg/ml for Cis, and 260.0-4400.0 nM (mean 2555.0 nM) for Tax. Selection pressures during treatment may contribute to drug resistance.     

Role of p53 in the induction of cyclooxygenase-2 by cisplatin or paclitaxel in non-small cell lung cancer cell lines

Non-small cell lung Cancer (NSCLC) is extremely resistant to chemotherapeutic agents, such as cisplatin. High expression of the inflammatory enzyme Cyclooxygenase-2 (COX-2) has been shown to inhibit chemotherapy-induced apoptosis, but little is known about COX-2 regulation upon drug treatment. Recent data indicate the tumor suppressor protein p53 as an important regulator of COX-2. Therefore, TP53 status could change tumor sensitivity to chemotherapy through induction of the anti-apoptotic protein COX-2. The main objective of this work was to analyze the effect of chemotherapy on the expression of COX-2, according to TP53 status. We report herein that lung cancer cell lines expressing wild-type p53, when exposed to cisplatin treatment, induced COX-2 (mRNA and protein), with concurrent synthesis of prostaglandins (PGE₂). In contrast, COX-2 expression was not changed after cisplatin treatment of cells containing an inactive form of p53. Further, after silencing of wild-type p53 expressed in A549 cells by RNA interference, cisplatin was no longer able to induce COX-2 expression. Therefore, we suggest that induction of COX-2 by cisplatin in NSCLC cell lines is dependent on p53. For paclitaxel treatment, an increase in COX-2 mRNA expression was observed in H460 and A549 (wild-type p53 cell lines). Moreover, paclitaxel treatment increased COX-2 expression in ACC-LC-319 cell lines (p53 null), showing a p53-independent effect. These data may have therapeutic implications in the selection of patients and strategy for future COX-2 inhibition trials.     

重组人 p53 腺病毒在晚期卵巢癌治疗中临床疗效及安全性评价简

目的：探讨重组人p53腺病毒注射液（rAd p53）联合TP／TC静脉化疗方案在晚期卵巢癌患者中的应用价值。方法：47例晚期卵巢癌术后患者,25例对照组患者单纯接受3周一次的TP／TC静脉化疗,共6—8疗程;22例实验组患者在化疗基础上加用2疗程rAd p53,比较2组患者近期、远期疗效及药物的安全性。结果：实验组CA125下降曲线显于对照组（P=0．037）;实验组无瘤生存期优于对照组（30．4个月vs21．5个月,P=0．012）;临床缓解率两组无差异（68．2％vs48．0％,P=0．135）;总生存期无差异（39．6个月vs32．5个月,P=0．13）。未发现rAd p53的严重不良反应。结论：rAd p53对于晚期卵巢癌患者的治疗有积极的作用,可以被很好的耐受,但结果尚需要进一步在临床应用中得以验证。     

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。     

重组人p53腺病毒联合肿瘤坏死因子对乳腺癌Ca761细胞的抑制作用

目的：探讨重组人p53腺病毒注射液（rAd/p53）联合肿瘤坏死因子（TNF）对荷瘤小鼠体内外的乳腺癌细胞Ca761的抑制效果。方法：MTT法,流式细胞术检测细胞凋亡率,观察rAd/p53和TNF对乳腺癌细胞的体外抑制作用及促凋亡作用。建立乳腺癌Ca761细胞小鼠移植瘤模型,瘤内分别注射rAd/p53和TNF及两药联合,观察对肿瘤生长的影响,并对肿瘤组织进行病理学和免疫组化检查。结果：rAd/p53和TNF体内外均可显著抑制乳腺癌Ca761细胞的生长,可以促进乳腺癌细胞Ca761株的凋亡。两药联合对肿瘤细胞具有协同杀伤作用。结论：重组人p53腺病毒注射液（rAd/p53）治疗Ca761乳腺癌细胞移植瘤是有效的,TNF也可抑制其生长,rAd/p53和TNF联合应用有协同作用,为临床乳腺癌的综合治疗提供了依据。     

Adenoviral-mediated p53 transgene expression sensitizes both wild-type and null p53 prostate cancer cells in vitro to radiation

Purpose/Objective: The effect of adenoviral-mediated p53 transgene expression on the radiation response of two human prostate cancer cell lines, the p53^wild-type LNCaP and p53^null PC3 lines, was examined. The objective was to determine if this vector sensitizes cells to radiation independently of their p53 status.

Methods and Materials: A recombinant adenovirus-5 vector (RPR/INGN 201, Introgen Therapeutics, Houston, TX) containing a CMV promoter and wild-type p53-cDNA (Ad5-p53) was used to facilitate p53 transgene expression. A multiplicity of infection (MOI) of 10–40 viral particles per cell was used, based on Ad5/CMV/lacz infection and staining for the β-galactosidase reporter gene product. Clonogenic assays were performed to evaluate the degree of sensitization to radiation of viral-transduced cells compared with irradiated nontransduced controls. The relative efficacy of these treatments to induce apoptotic cell death was determined using the TUNEL assay.

Results: The delivery of Ad5-p53 (10 MOI) reduced control plating efficiency from 36.5% to 0.86% in the LNCaP cell line and from 75.1% to 4.1% in the PC3 cell line. After correcting for the effect of Ad5-p53 on plating efficiency, the surviving fraction after 2 Gy (SF2) of gamma-irradiation was reduced over 2.5-fold, from 0.187 to 0.072, with transgene p53 expression in the LNCaP cell line. Surviving fraction after 4 Gy (SF4) was reduced over 4.5-fold, from 0.014 to 0.003, after Ad5-p53 treatment. In the PC3 cell line, Ad5-p53 (40 MOI) reduced SF2 over 1.9-fold from 0.708 to 0.367, and SF4 over 6-fold from 0.335 to 0.056. In both the LNCaP and PC3 cell lines, the combination of Ad5-p53 plus radiation (2 Gy) resulted in supra-additive apoptosis (20% for LNCaP and 15% for PC3 at 50 MOI), above that seen from the addition of the controls; control vector Ad5-pA plus RT (0.15% for LNCaP and 1.44% for PC3), Ad5-p53 alone (28.6% for LNCaP and 21.7% for PC3), RT alone (0% for LNCaP and 0.23% for PC3), or Ad5-pA alone (0.1% for LNCaP and 0.29% for PC3).

Conclusion: The clonogenic survival and apoptosis data demonstrate that p53 transgene expression sensitizes human prostate adenocarcinoma cells in vitro to irradiation. As this effect was observed in both the p53^wild-type LNCaP and p53^null PC3 lines, radiosensitization was independent of p53 status.     

重组p53腺病毒联合中药治疗小鼠腹水瘤的疗效

目的： 〖HT5"SS〗探讨p53腺病毒（简称Adp53）注射液联合中药得力生及消癌平治疗小鼠腹水瘤的协同作用。〖HT5W〗方法：〖HT5"SS〗建立昆明小鼠H22肝癌腹水瘤模型,建模48 h后分组分别给予Adp53、中药得力生和消癌平或两药联用。用药1周后检测体重、腹围、腹水量、腹水瘤细胞计数,流式细胞仪测定瘤细胞凋亡百分率以及细胞周期情况。〖HT5W〗结果：〖HT5"SS〗 Adp53、得力生、消癌平单药治疗组,除体重以外的上述指标与对照组比较均有统计学差异（P＜0.05）,Adp53+得力生和Adp53＋消癌平组的腹围增长率、腹水量、瘤细胞计数、晚期细胞凋亡率均明显少于各单药治疗组（P＜0.05）。 〖HT5W〗结论： 〖HT5"SS〗腹腔注射Adp53、得力生和消癌平均可抑制H22细胞生长并减少腹水形成,Adp53与中药得力生、消癌平联合腹腔内给药对治疗腹水瘤有协同作用。