p53基因对胶质瘤细胞系U251生长抑制作用的研究

背景与目的：肿瘤抑制基因p53是调节多种与细胞周期、凋亡、DNA修复等有关基因表达的转录因子。p53基因在大约30％的胶质瘤中发生突变，在胶质瘤的发生和发展中起重要作用。本文主要探讨野生型p53基因过表达对脑胶质瘤细胞系U251细胞生长抑制的机制。方法：通过p53腺病毒表达载体pAdCMV-p53及空载体pAdCMV-lacZ分别感染U251细胞系，RT-PCR及Westem blot方法检测转染效率；并通过MTT检测生长抑制率、流式细胞仪检测细胞周期及TUNEL检测分析细胞凋亡等指标观察p53基因对U251细胞生长的影响。结果：MOI为100时，野生型p53基因的过表达可引起U251细胞G0、G1期阻滞、诱导U251细胞凋亡以及引起U251细胞生长抑制。结论：p53基因可以通过细胞周期G0、G1期阻滞及诱导细胞凋亡抑制胶质瘤细胞系U251的生长。     

野生型p53基因与双脱水二乙酰卫矛醇联合诱导肝癌HLE细胞凋亡的研究

背景和目的：p53基因是抑癌基因,研究发现在人类肿瘤中p53基因常发生突变。实验证明野生型p53基因不但抑制肿瘤细胞增殖,而且诱导肿瘤细胞凋亡。但是,单独应用野生型p53基因诱导肿瘤细胞凋亡的作用不很明显。因此,野生型p53基因与药物的联合作用来提高肿瘤细胞凋亡率是目前对肿瘤研究的一新领域。本实验通过转染野生型p53基因联合双脱水二乙酰卫矛醇(1,2：5,6-dianhydro-3,4-diacetylgalactitol,DADAG)作用于肝癌细胞株HLE,研究联合诱导肿瘤细胞的凋亡作用。方法：野生型p53基因通过脂质体Lipofectamine介导转染人肝癌细胞株HLE,同时加入DADAG。96h后,用流式细胞仪、DNA电泳方法进行细胞凋亡分析。结果：流式细胞仪分析细胞凋亡结果：阴性对照组为1．4％、转染pUHD10-3组为3．5％、DADAG处理组为32．6％、转染pUHD10-3-p53组为43．4％、转染p53基因及DADAG处理组为74．6％。DNA电泳发现,转染p53基因及DADAG处理组有到DNA梯形条带。结论：野生型p53基因与DADAG均可诱导人肝癌细胞HLE发生凋亡,转染野生型p53基因与DADAG联合作用,有促进肝癌细胞凋亡的作用。     

p53基因对人胚肺成纤维细胞周期影响的研究

背景与目的:研究野生型和179位残基突变型p53基因在HELF细胞周期调控中的作用.探讨p53基因的179位残基突变对细胞生长的影响.材料与方法:用野生型p53(pcDNA3-wtp53)和179位残基突变的突变型p53(pcDNA3-mtp53)转染HELF细胞,观察细胞生长情况,绘制细胞生长曲线;用流式细胞仪分析细胞周期;用RT-PCR和Western blotting方法检测p53基因转染后HELF细胞周期相关基因mRNA和蛋白的表达.结果:野生型p53表达的上调使HELF细胞周期阻滞于G1期,细胞体积减小,并下调cyclin D3、cyclin E、Cdk2和Cdk4的表达,同时上调p21的表达.而179位残基突变的突变型p53表达的上调则促进细胞周期从G1期到S期的转换,同时细胞体积增大,上调cyclin A和Cdk4的表达.结论:p53的179位残基突变对于HELF细胞cyclin A和Cdk4的表达有诱导作用,并可能借此促进细胞周期进程.     

p53基因诱导胱癌细胞HTB9凋亡及相关基因的表达

目的 研究野生型p53基因诱导膀胱癌细胞凋亡的作用,及其对凋亡相关基因bcl-2、bax和ICE表达的调控。方法 将野生型p53基因重组腺病毒载体转染人膀胱癌细胞HTB9,应用RT－PCR检测bax的mRNA表达水平,应用免疫组化法检测bcl-2、bax和ICE蛋白表达水平,以DNA琼脂糖凝胶电泳、脱氧核苷酸转移酶介导的dUTP切口末端标记技术（TUNEL）和流式细胞仪检测细胞凋亡。结果 野生型p53基因导入可诱导HTB9细胞凋亡,凋亡细胞百分率可达50．4％;bax mRNA和蛋白水平增高,ICE和Bcl-2蛋白水平分别增高和下降。结论 野生型p53基因很可能是通过调控凋亡相关基因ICE、Bax和Bcl-2的表达来诱导细胞凋亡。     

野生型p53对白血病K562细胞中心体过度复制的抑制作用

背景与目的：肿瘤组织细胞中p53基因突变或缺失是导致非整倍体的发生和基因组不稳定的主要原因之一：最近研究发现慢性粒细胞白血病（chronic myelogenous leukemia．CML）各期患者均有中心体异常,且异常的程度与临床分期有关。急变期显示更为严重的中心体异常。本研究建立携野生型p53基因的CML急变K562细胞株．以研究该细胞内野生型p53基因表达后p53信号转导通路对K562细胞中心体的影响。方法：用HEK293细胞扩增重组p53野生型、突变型及空载腺病毒载体,联合polybrene分别感染K562细胞;未接受感染的细胞作为空白对照。流式细胞术检测重组腺病毒载体感染效率,Western blot检测P53蛋白表达。间接免疫荧光染色后用激光共聚焦计数K562细胞中心体的变化。Westernblot检测p53信号转导通路下游效应分子生长阻滞和DNA损伤应答基因Gadd45a（growtharrest and DNA damage）、BubRl（Bublrelated）、AuroraA的表达。结果：成功建立携野生型p53基因的K562细胞株．腺病毒载体感染效率达60％以上,野生型p53可在K562细胞中持续表达。感染72h后,携野生型p53基因的K562细胞中中心体数量异常（n〉2）的细胞比例降至（0．38＋0．02）％,与空白对照组（0．71＋0．14）％比较其差异有统计学意义（P〈0．05）;Westernblot结果发现p53信号转导通路下游效应分子Gadd45a、BubRl表达分别上调93％、88％．而AuroraA的表达下降56％（P值均〈0．05）。结论：重组腺病毒介导的野生型p53基因能够在白血病K562细胞中持续表达：野生型P53蛋白可能通过转录激活-依赖途径上调Gadd45a、BubR1表达以及转录激活-非依赖途径使AuroraA的表达下降,从而抑制K562细胞中心体的过度复制。     

二乙酰去水卫矛醇联合野生型p53基因诱导肝癌BEL-7404细胞凋亡的作用

目的 实验通过转染野生型p53基因与应用二乙酰去水卫矛醇(1，2：5，6-Dianhydro-3，4-diacetylgalactitol，DADAG)联合作用于肝癌BEL-7404细胞，来研究p53基因联合二乙酰去水卫矛醇诱导肝癌BEL-7404细胞的凋亡作用。方法野生型p53基因通过脂质体Lipofectamine介导转染人肝癌BEL-7404细胞，同时加入DADAG作用。96h后，用流式细胞仪进行细胞凋亡分析。结果流式细胞仪分析细胞凋亡结果：阴性对照组为1．6％、转染pUHD10-3组为3．9％、用DADAG处理组为29.4％、转染pUHD10-3-p53组为35.3％、转染p53基因及用DADAG处理组为59．8％。结论野生型p53基因与DADAG均可诱导人肝癌BEL-7404细胞发生凋亡，转染野生型p53基因与DADAG联合作用，有促进肝癌BEL-7404细胞凋亡的作用。     

乳腺癌血清p53抗体的临床意义及其与其他生物学参数间的关系

p53是一种抑癌基因，与细胞DNA修复、衰老、分化、凋亡和细胞周期的阻滞密切相关。野生型p53的缺失可导致基因的不稳定和细胞的恶性转化。有证据表明，p53突变会导致细胞内突变型p53的积聚，同时可以在患者血中检测到p53抗体。p53抗体在美国及欧洲妇女乳腺癌中出现率为15％～     

曲古抑菌素A通过p53转录激活途径诱导尤文肉瘤细胞凋亡

目的：探讨组蛋白去乙酰化酶抑制剂曲古抑菌素A（trichostatin A,TSA）诱导尤文肉瘤细胞株WE-68和VH-64凋亡及作用机制.方法：四甲基偶氮唑蓝法（MTT法）测定细胞增殖抑制率.流式细胞计数法测量TSA给药后细胞周期中sub-G1含量的变化.免疫印迹法（Western-blot）检测细胞中活化型多聚ADP核糖多聚酶（cleaved-PARP）,p53-lys382残基乙酰化和p53蛋白总量的表达.实时定量PCR和siRNA转染技术测定p53多个下游基因的mRNA水平改变和p53表达下调对TSA诱导凋亡的影响.结果：TSA抑制了尤文肉瘤细胞的增殖,诱导细胞周期中sub-G1含量和凋亡终产物cleaved-PARP蛋白表达的增加.TSA给药后,p53-lys382残基乙酰化表达量呈浓度依存性增加,同时上调p53下游因子p21,mdm2,Bax和PUMA的mRNA水平.另一方面,p53蛋白表达的下调明显削弱了TSA介导的p21表达的上调和cleaved-PARP的产生.结论：组蛋白去乙酰化酶抑制剂TSA能够通过激活p53高乙酰化表达来恢复p53转录功能,从而诱导尤文肉瘤细胞株产生凋亡.     

氧化铁磁性纳米颗粒介导wt-p53基因对肺腺癌细胞增殖和凋亡的影响

目的：探讨氧化铁磁性纳米颗粒介导野生型p53基因（wild type p53,wt-p53）对耐顺铂人肺腺癌细胞A549/DDP增殖抑制和凋亡诱导的作用。方法：氧化铁磁性纳米颗粒介导wt-p53转染肺腺癌细胞A549/DDP作为实验组,以纳米颗粒介导空载体pcDNA3转染作为阴性对照组,脂质体介导wt-p53转染作阳性对照组。MTT法和绘制生长曲线观察基因转染对A549/DDP细胞增殖抑制作用,荧光显微镜、流式细胞术观察其对A549/DDP细胞诱导凋亡作用,RT-PCR检测其对A549/DDP细胞Bax mRNA表达的影响。结果：氧化铁磁性纳米颗粒介导wt-p53对人肺腺癌细胞A549/DDP增殖有持续的抑制作用,而以脂质体介导wt-p53对增殖抑制作用持续时间短暂;纳米颗粒介导wt-p53对人肺腺癌细胞A549/DDP诱导凋亡作用明显强于以脂质体载体;同时介导wt-p53上调Bax mRNA表达水平的作用也明显强于以脂质体载体。结论：氧化铁磁性纳米颗粒介导wt-p53转染对人肺腺癌细胞A549/DDP有持续的增殖抑制和诱导凋亡的作用。     

p53转染对肝癌细胞系Hep3B的作用

目的：研究野生型和突变型p５３基因cＤＮＡ转染对肝癌细胞系Ｈep３Ｂ的作用。方法：通过采用脂质体介导转染技术,将野生型、突变型p５３的真核表达重组质空载体质粒分别导 入一种p５３和Ｒb基因缺失的肝癌细胞系Ｈep３Ｂ。结果：经Ｇ４１８筛选获得稳定的整合了野生型p５３的克隆（wide-type p53, wt-p53)、突变型p53细胞表达p53细胞的p２１＾wafl/cipl蛋白表达水平升高。wt-p53细胞生长较ＴＤＮ Ｐ５３、pＮeo细胞缓慢,但不出     

Implication of p53 in growth arrest and apoptosis induced by the synthetic retinoid CD437 in human lung cancer cells.

CD437 is a novel retinoid that can induce apoptosis in a variety of tumor cell types by an unknown mechanism. We found that CD437 up-regulated the expression of p21(WAF1/CIP1), Bax, and Killer/DR5 and induced G1 arrest and rapid apoptosis in three human non-small cell lung carcinoma cell lines with wild-type p53 but not in five cell lines with mutant p53, suggesting a role for p53 in the effects of CD437. Using H460 cells in which wild-type p53 protein was degraded by transfection of the human papillomavirus 16 E6 (HPV-16 E6) gene and H460 cells transfected with a control plasmid only, we found that CD437 increased p53, p21(WAF1/CIP1), Bax, and Killer/DR5 in the control transfectants. In contrast, the constitutive p53 protein level was suppressed, and the ability of CD437 to increase p53 and its downstream genes was compromised in E6 transfectants. In addition, CD437 induced G1 arrest and apoptosis in the control transfectants but not in the E6-transfected cells. These results indicate that p53 plays a role in CD437-induced growth inhibition and apoptosis in human non-small cell lung carcinoma cells.     

Delay of dimethylbenz[a]anthracene-induced mammary tumorigenesis in transgenic mice by apoptosis induced by an unusual mutant p53 protein

Murine p53 containing an Arg → Leu substitution at amino acid 172 possesses many properties characteristic of wild-type p53, including the ability to induce p21/WAF/Cip1 and apoptosis. To determine if p53–dependent apoptosis plays a critical role in mammary tumorigenesis, transgenic mice were generated in which the expression of this mutant p53 protein was targeted to the mammary gland by using the rat whey acidic protein gene promoter. Mice bearing pituitary isografts were treated with 7,12–dimethylbenz[a]anthracene (a) and examined for mammary tumor development. Mice overexpressing the p53 transgene exhibited a statistically significant increase in apoptosis in the mammary gland and a statistically significant decrease in the incidence of DMBA-induced mammary tumors. No difference in tumor incidence was observed in mice without pituitary isografts who were treated with DMBA, because the transgene is not overexpressed in the absence of hormone stimulation provided by the pituitary isograft. The unexpected wild-type properties of the 172^Arg→Leu mutant p53, including its ability to stimulate apoptosis, make it a possible candidate for use in gene therapy protocols. ©1995 Wiley-Liss, Inc.     

The mutant p53-conformation modifying drug,CP-31398, can induce apoptosis of human cancer cells and can stabilize wild-type p53 protein

CP-31398, a styrylquinazoline, emerged from a screen for therapeutic agents that restore a wild-type DNA-binding conformation of mutant p53 to suppress tumors in-vivo (Science 286, 2507, 1999). We investigated the growth inhibitory mechanism of CP-31398 using nine human cancer cell lines containing wild-type, mutant or no p53 expression. Six of nine cell lines underwent apoptosis after exposure to CP-31398, while two cell lines, DLD1 colon cancer and H460 lung cancer, underwent exclusively cell cycle arrest. Cell cycle arrest preceded the apoptosis in some cases. CP-31398 did not inhibit growth of the p53 non-expressing ovarian cancer cell line SKOV3. Interestingly, we found that wild-type p53 protein is stabilized upon CP-31398 exposure. p53 target genes such as p21WAF1/Cip1, and KILLER/DR5 were upregulated by CP-31398, but their expression did not correlate with arrest or apoptosis induction. Combination of CP-31398 and TRAIL or chemotherapeutic agents enhanced cancer cell killing effect possibly through upregulation of p53-regulated genes such as KILLER/DR5. Bax-/-, wild-type p53-expressing cells displayed reduced susceptibility to killing by CP-31398. An Affymetrix GeneChip Array screen revealed that CP-31398 alters expression of non-p53 target genes in addition to p53-responsive genes. Although our preliminary data suggest that CP-31398 does not alter wild-type p53:MDM2 interaction, further efforts are required to elucidate the mechanism of wild-type p53 stabilization by CP-31398. The results increase our understanding of CP-31398 action, and suggest strategies for improving its specificity, possibly through use of microarrays to screen related compounds with higher mutant p53-specificity.     

Changes in p21WAF1, pRb, Mdm-2, Bax and Bcl-2 expression in cervical cancer cell lines transfected with a p53 expressing adenovirus

The aim of this study was to provide some insights into the molecular mechanisms involved in p53-dependent apoptosis and growth arrest. Changes in the levels of p53 protein and proteins regulated by p53 were studied in relation to events of the cell cycle and apoptosis in cervical cancer cell lines upon transfection with a p53 expressing adenovirus (Ad5-p53). The post-transfection level of p53 protein in SiHa cells was found to be unchanged during the 24-48 h period. In contrast, the level of p21WAF1 protein was shown to increase to its highest level at 24 h, and decreased gradually up to 48 h after the Ad5-p53 transfection. We further noted that the increase of p21WAF1 was accompanied by G1 arrest at 24 h and the decrease of p21WAF1 was associated with apoptosis at 36-48 h after transfection. An anti-p21WAF1 antibody cross-reactive protein band of approximately 14 kDa was observed in HeLa and C-33A cells when these cells were committed to apoptosis upon Ad5-p53 transfection. In SiHa cells, phosphorylation of pRb was inhibited during the early stage of Ad5-p53 transfection. This was followed by the cleavage of pRb. However, Ad5-p53 transfection did not change the levels of Bax and Bcl-2 proteins. Our results suggested that, Bax and Bcl-2 may not be important for the apoptosis of these cells, whereas cleavage of Rb, and the decrease of p21WAF1 could play important roles in p53-dependent apoptosis.     

p33ING1b增强骨肉瘤细胞U2OS对足叶乙甙的敏感性

背景与目的作为一个新的抑癌基因,ING1与p53有许多相似的生物学功能,如细胞生长抑制、DNA修复、凋亡和化疗敏感性等.本实验目的在于研究p33ING1b对骨肉瘤细胞药物敏感性的影响并探讨其作用机制.方法瞬时转染p33ING1b进入骨肉瘤细胞株U2OS后,用足叶乙甙(etoposide,VP-16)处理24 h,然后采用台盼蓝拒染法计数活细胞数并计算细胞生长抑制率,流式细胞仪、DAPI染色等方法检测细胞凋亡率,Western blot技术检测p53、p21WAF1、MDM2和Bax蛋白的表达.结果台盼蓝染色结果表明,瞬时转染p33ING1b进入骨肉瘤细胞株U2OS后,再用VP-16处理24 h,细胞生长抑制率明显增加[(63.1±5.1)%].流式细胞计数和DAPI染色检测结果表明,细胞凋亡率明显增加(62.7%).Western blot检测结果显示,外源性p33 ING1b高表达明显提高了p53及其下游基因p21WAF1和Bax蛋白的表达水平,转染p33ING1b进入骨肉瘤细胞株U2OS后,再用VP-16处理24 h,p53、p21和Bax蛋白表达增加.在各实验组中,MDM2的蛋白表达没有明显变化.结论p33ING1b能够上调p53蛋白,并上调p53下游因子--p21WAF1和Bax的表达水平,通过p53依赖性凋亡信号通路,提高骨肉瘤细胞U2OS对VP-16的敏感性.     

Effects of p53 mutations on cellular sensitivity to ionizing radiation

Mutations in the p53 tumor suppressor gene have been found in more than 50% of human tumors including those in breast, colon, lung, and oral cavity. However, the significance of p53 mutation in radiation sensitivity and its underlying mechanisms still remains unclear. In this study, we have measured the effects of p53 mutation on cell cycle delay, apoptosis, and radiation sensitivity using mouse cells transfected with different forms of p53 mutations. Wild-type p53 and p53-Null mouse embryo fibroblast cells were used as positive and negative controls, respectively. Exponentially growing cells were irradiated with 0- to 9-Gy gamma rays and then assayed for cell survival, p53 expression, cell cycle checkpoint, and apoptosis. Cell survivals determined by clonogenic assay show that p53 mutant cells are generally more sensitive to ionizing radiation than cells with wild-type p53. Western blot analysis indicates that exposure to 6-Gy gamma rays increases the p53 expression levels by two- to threefold in wild-type p53 cells. However, the p53 level remains unchanged in cells with mutant p53 during the same postirradiation period. Irradiation with 6-Gy gamma rays produces G2/M arrest in all cell lines, indicating that p53 is probably not involved in the G2/M checkpoint. However, all mutant cells fail to show any significant G1/S arrest after irradiation, suggesting that G1/S arrest may be implicated in radiation sensitivity. Finally, there is very little apoptosis (<3% by Tat-mediated dUTP nick-end labeling [TUNNEL] and morphologic assays) detected in wild-type and p53 mutant cell lines after 6-Gy gamma rays. Our results suggest that mutant forms of p53 represent a phenotype that affects the radiation sensitivity and is not dependent on the apoptotic pathway.     

p73 can suppress the proliferation of cells that express mutant p53

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer. p73, a member of the p53 family, has been found to exhibit activity similar to that of p53, including the ability to induce growth arrest and apoptosis. p53 and p73 have a high percentage of similarity at several domains, including the DNA binding domain. This domain in p53 is the location of missense mutations in many human cancers. Mutant p53, which cannot suppress cell proliferation, has been found to have a dominant-negative activity that inactivates wild-type p53. To determine the effects of mutant p53 on wild-type p73, we have established cell lines expressing both mutant p53 and wild-type p73 in a dual-inducible system. This system expresses mutant p53 in a tetracycline-repressible system and p73beta in an ecdysone-inducible system in a p53-null lung carcinoma parental cell line. We have found that wild-type p73beta, in the presence of mutant p53, retains the ability to transactivate p21 and suppresses cell growth through induction of both cell cycle arrest and apoptosis. In addition, in cell lines expressing wild-type p53 and wild-type p73beta, we have found that these proteins cooperate to additively transactivate p21 and suppress cell proliferation.     

Antisense Bcl-xl down-regulation switches the response to topoisomerase I inhibition from senescence to apoptosis in colorectal cancer cells, enhancing global cytotoxicity.

PURPOSE: To identify determinants of the effect of antisense-mediated Bcl-xl down-regulation (Bcl-xl knockdown) on the response of colorectal cancer cells to SN38, the active metabolite of irinotecan, a topoisomerase I inhibitor licensed for colorectal cancer chemotherapy. EXPERIMENTAL DESIGN: Using wild-type HCT116, p53 null, Bax null, or p21/WAF1 null isogenic derivatives, we measured expression of regulators of cellular response, and associated growth arrests or apoptosis, after SN38 treatment, with or without antisense-mediated Bcl-xl knockdown. RESULTS: A modified phosphorothioate antisense oligonucleotide (ISIS15999) reduced Bcl-xl protein expression by approximately 90%. SN38 induced p53, Bax, Bcl-xl, and p53-dependent p21/WAF1 protein accumulation. The Bax:Bcl-xl ratio changed little. In wild-type HCT116, but not in Bax null cells, Bcl-xl knockdown induced a shift in response from drug-induced senescence to apoptosis, and enhanced the global cytotoxicity of SN38. In p53 null or p21/WAF1 null cells marked apoptosis occurred after SN38 alone, and was additionally enhanced by Bcl-xl knockdown in p21/WAF1 null cells but not in p53 null cells. CONCLUSIONS: Drug-induced senescence is associated with late relapse after therapy in transgenic models of cancer in vivo. We have shown that abolition of p21/WAF1-mediated drug-induced senescence or antisense-mediated Bcl-xl knockdown can both, independently, enhance the apoptotic response of colorectal cancer cells to SN38 in vitro. The growth arrest suppresses a p53-independent apoptotic pathway, whereas Bcl-xl induction suppresses a p53 and Bax-dependent apoptotic pathway. The combination of irinotecan and Bcl-xL antisense merits testing in models of colorectal cancer in vivo.