SMMC-7721肝癌细胞系中p53与乙型肝炎病毒相互作用的研究

目的 进一步观察乙型肝炎病毒(HBV)与p53的作用关系，以便更深入地了解HBV对原发性肝细胞癌发生的影响。方法 选用SMMC-7721肝癌细胞系(表达野生型p53，HBV阴性)为靶细胞，采用脂质体转染法，将pCMVp53单独或者与野生型HBV质粒(pCMVHBVa)、变异型HBV质粒(pCMVHBVb)共转染细胞，用annexin V-FITC标记及流式细胞仪检测细胞凋亡的变化；各组分别共转染报告基因：PG13-CAT，含有p21基因启动子的p21-LUC，通过CAT酶以及荧光素酶的检测，观察p53活性情况以及对p21启动子的活化情况；western blot方法检测各转染组p53蛋白表达情况。结果 单独的pCMVp53质粒转染SMMC-7721细胞系能够促进p21报告基因的表达，细胞凋亡率升高；HBVa质粒与pCMVp53共同转染可使细胞中p53蛋白表达水平，p53对报告基因PG13-CAT的活化作用以及对p21基因启动子的激活作用增强，细胞凋亡率增加，细胞生长受到抑制，而与pCMVHBVb共转染时则不能。结论 HBV在细胞内的复制可能通过使p53蛋白半衰期延长，及p53蛋白的表达及作用增强，促进p53下游基因p21的转录，导致细胞凋亡率增强，细胞生长受到抑制;p53可能通过某种机制对HBV的复制具有一定的促进作用。     

p53在神经酰胺诱导内皮细胞凋亡中的作用

目的:探讨p53在外源性神经酰胺(C2-ceramide)诱导人脐静脉内皮细胞凋亡中的作用。方法:体外培养脐静脉内皮细胞,用不同浓度的C2-ceramide进行处理,Tunel检测细胞凋亡;逆转录-聚合酶链反应(RT-PCR)和Western blot检测p53的表达;并用p53的抑制剂PFT-α(pifithrin alpha,PFT-α)预处理细胞,再加入C2-ceramide与对照组比较观察细胞凋亡指数。结果:C2-ceramide呈剂量依赖性诱导内皮细胞凋亡;p53的表达随着C2-ceramide浓度的增加呈现降低的趋势;PFT-α不能抑制C2-ceramide诱导的凋亡。结论:神经酰胺能诱导内皮细胞的凋亡,可能不依赖于p53途径。     

老年急性心肌梗死猝死者心肌细胞凋亡与p53和Bcl—2蛋白表达的相关性研究

目的探讨p53和Bcl-2蛋白在老年急性心肌梗死(AMI)猝死者心肌细胞凋亡中的作用.方法用TUNEL法检测15例猝死者和10例心脏正常的车祸死亡者的心肌细胞凋亡,用免疫组化法检测p53和Bcl-2蛋白在心肌中的表达水平,并探讨它们之间的相互关系.结果 TUNEL法发现,猝死者梗死区的心肌细胞凋亡千分数老年组(663.00±117.12‰)明显高于正常对照(34.30±20.68‰)(P=0.00);p53蛋白表达阳性千分率(462.12±237.58‰)明显高于正常对照者(39.45±24.67‰)(P=0.000),且与梗死区心肌细胞凋亡数成明显的正相关性(r为0.701,P＜0.005);Bcl-2蛋白表达阳性率(40.83±31.29‰)明显低于正常对照(120.76±66.67‰)(P=0.0037),且与梗死区心肌细胞凋亡数成明显的负相关性(r为-0.840,P＜0.001);梗死区心肌细胞内p53和Bcl-2蛋白表达数之间呈明显的负相关性(r=-0.829,P＜0.001).结论 AMI猝死者梗死区心肌细胞存在明显的凋亡现象,且受p53蛋白上调、Bcl-2蛋白下调,两者相反调节其凋亡.     

Contributions of p53 and PMA to g-irradiation induced apoptosis in Jurkat cells

Several mutations prevent the expression of p53 in the human lymphoblastoid T cell line Jurkat. Restoration of p53 in Jurkat cells had no effect on the cell growth but markedly increased the amount of apoptosis induced by g-irradiation. Inhibition of RNA synthesis using 5,6-dichlorobenimidizole riboside had little effect on apoptosis induced by irradiation in the presence of p53 and did not affect the p53-independent apoptotic pathway. Expression of p53 also had no effect on the expression levels of proteins such as Fas, GADD45, Bax, Bcl-2, Bcl-x_L or p53 induced proteins (PIGS) in resting cells or after irradiation. Activation of protein kinase C by phorbol 12-myristate 13-acetate produced an almost complete inhibition of p53-independent apoptosis following irradiation, whereas no significant effect was observed on the rate of p53-induced apoptosis. Although phorbol 12-myristate 13-acetate strongly induced p21 and stabilised p53 in the resting transfected Jurkat cells, neither apoptosis nor cell arrest was observed. In summary, this work shows that p53 enhances the radiosensitivity of Jurkat cells through an apoptotic process that is triggered by irradiation and is largely independent of RNA synthesis and protein kinase C activation. Apoptosis in p53- negative Jurkat cells is strongly inhibited by PMA indicating that the pathway triggered by p53 may be distinct from apoptotic pathways used in its absence.     

The role of XPB in cell apoptosis and viability and its relationship with p53, p21 and c-myc in hepatoma cells

BACKGROUND: Accumulation of DNA damage has been implicated in hepatocarcinogenesis. XPB plays a pivotal part in repairing damaged DNA. However, up to now, the biological effect of XPB on hepatoma cells remains elusive. MATERIALS AND METHODS: Here, we investigated the role of XPB in the apoptosis and the viability of hepatoma cells by using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labelling and cell viability assay; we also investigated their relationship with p53, p21(waf1/cip1) and c-myc by using the RT-PCR and Western blot. RESULTS: Compared with the control cells HepG2/pcDNA3.1 or HepG2, XPB-transfected HepG2 cells (HepG2/pcDNA3.1-XPB) displayed lower viability, weaker activity and higher apoptosis index. At the same time, an increased expression of p21(waf1/cip1) mRNA, protein and p53 protein in addition to a decreased expression of c-myc mRNA and protein were detected in HepG2/pcDNA3.1-XPB cells. CONCLUSIONS: Our results indicated that XPB could inhibit the proliferation of hepatoma cells and had a positive effect on the expression of p53 and p21(waf1/cip1) but a negative effect on c-myc.     

PTEN在HepG2细胞中诱导凋亡发生及上调p53表达

目的 研究肿瘤抑制基因PTEN对HepG2细胞凋亡及p53蛋白表达的影响,并探讨相关机制。方法 将携带有野生型PTEN基因及突变型G129E-PTEN,C124A-PTEN基因的真核表达载体转染HepG2细胞,利用western印迹杂交检测PTEN表达、蛋白激酶B(PKB/Akt)和焦点粘附激酶(FAK)的磷酸化状态,以及野生型p53蛋白表达水平的变化,并应用流式细胞仪,激光共聚焦技术检测细胞周期及细胞凋亡情况。结果 与对照细胞相比,转染野生型PTEN和G129E-PTEN的HepG2细胞中磷酸化FAK(-65％,-65％)与磷酸化Akt(-93％,-35％)表达均存在不同程度的下调,而细胞凋亡率分别增加至19.8％±1.2％和9.2％±0.6％,并且p53蛋白表达上调( 120%,, 50％);然而转染C124A-PTEN的细胞中各项检测指标均无明显变化。 结论 PTEN依赖其蛋白磷酸酶活性抑制FAK的磷酸化;并主要通过脂质磷酸酶活性抑制Akt的磷酸化,并诱导HepG2细胞凋亡和p53蛋白表达上调。     

Down-expression of tumor protein p53-induced nuclear protein 1 in human gastric cancer

AIM: Overexpression of tumor protein p53-induced nuclear protein 1 (TP53INP1) induces G1 cell cycle arrest and increases p53-mediated apoptosis. To clarify the clinical importance of TP53INP1, we analyzed TP53INP1 and p53 expression in gastric cancer. METHODS: TP53INP1 and p53 expression were examined using immunohistochemistry in 142 cases of gastric cancer. The apoptosis of gastric cancer cells was analyzed using the TUNEL method. The relationship between the expression of TP53INP1 and clinicopathological factors was statistically analyzed. RESULTS: TP53INP1 was expressed in 98% (139/142 cases) of non-cancerous gastric tissues and was down-expressed in 64% (91/142 cases) of gastric cancer lesions from the same patients. TP53INP1 expression was significantly decreased (43.9%) in poorly differentiated adenocarcinoma compared with well or moderately differentiated adenocarcinoma (81.6%). Cancers invading the submucosa or deeper showed lower positively (59.1%) compared with mucosal cancers (85.2%). Decrease or loss of TP53INP1 expression was significantly correlated with lymphatic invasion (54.3% vs 82.0% without lymphatic invasion) and node-positive patients (31.3% vs 68.3% in node-negative patients). P53 was expressed in 68 (47.9%) patients of gastric cancer, whereas it was absent in normal gastric tissues. A significant association was also observed between TP53INP1 status and the level of apoptosis in tumor cells: the apoptotic index in TP53INP1-positive tissues was significantly higher than that in TP53INP1-negative portions. Finally, when survival data were analyzed, loss of TP53INP1 expression had a significant effect in predicting a poor prognosis (P=0.0006). CONCLUSION: TP53INP1-positive rate decreases with the progression of gastric cancer. TP53INP1 protein negativity is significantly associated with aggressive pathological phenotypes of gastric cancer. TP53INP1 is related to the apoptosis of gastric cancer cells. The decreased expression of the TP53INP1 protein may reflect the malignant grade of gastric cancer and is regarded as an adverse prognostic factor.     

Expression of p53 and Ki-67 antigen in bone marrow giant proerythroblasts associated with human parvovirus B19 infection

Giant proerythroblasts are hallmarks of human parvovirus B19 infection. We attempted to characterize these cells in 5 patients with parvovirus B19-induced pure red cell aplasia using immunostaining of paraffin-embedded bone marrow sections with antibodies against erythroid-lineage-specific proteins, viral capsid antigen VP-1, and apoptosis- and cell-cycle-related proteins. Giant proerythroblasts are immunohistochemically consistent with early erythroid precursors of cells in the differentiation stage of CD34-, cytoplasmic spectrin+, glycophorin A-, and band-3-. VP-1 was expressed in the nucleus and cytoplasm of small- to medium-sized spectrin+ erythroid cells but not in giant proerythroblasts. The giant proerythroblasts displayed nuclear staining for p53 (41%+/-16%) and Ki-67 antigen (100%+/-0%) and cytoplasmic staining for Bax (65%+/-11%) and procaspase-3 (78%+/-10%), whereas they were not stained for p21Wafl/Cip1, active form of caspase-3, or terminal deoxynucleotidyltransferase-mediated deoxyuridine nick-end labeling (TUNEL). Antiapoptotic proteins, Bcl-2 and Mcl-1, were not expressed in the giant cells, and Bcl-x was infrequently expressed in these cells (11%+/-4%). These immunohistochemical findings suggest that giant proerythroblasts are proliferating erythroid precursors with accumulation of nonfunctional p53.     

p53 allele deletion and protein accumulation occurs in the absence of p53 gene mutation in T-prolymphocytic leukaemia and Sezary syndrome

In a series of 24 patients with chronic T-lymphoid disorders [13 T-prolymphocytic leukaemia (T-PLL) and 11 Sezary syndrome] we have studied (i) chromosome 17p abnormalities and p53 allele deletion by fluorescence in situ hybridization; (ii) mutation in the exons of the p53 gene by direct DNA sequencing; and (iii) p53 protein expression by immunocytochemistry and, in some cases, also by flow cytometry with DO-1, a monoclonal antibody to the p53 protein. The study revealed p53 deletion and accumulation of p53 protein in the absence of mutation in the exons that included the hot-spots and differs from that described in B-prolymphocytic leukaemia. Seven T-PLL and five Sezary syndrome patients had p53 overexpression, and five T-PLL and nine Sezary syndrome patients showed p53 deletion. Although the majority of cases with p53 accumulation had p53 deletion, the proportion of cells with the deletion did not correlate with the proportion of cells positive for p53 expression. Two cases of T-PLL showed strong p53 expression in the absence of p53 deletion, and one case of Sezary syndrome with p53 deletion in 97% of cells did not express p53. These findings suggest that a non-mutational mechanism exists for the accumulation of p53 protein in these T-cell disorders. The oncogenic effect of the accumulating wild-type protein has been reported in other malignancies. Whether haploidy resulting from p53 deletion contributes to this mechanism has yet to be determined. Alternatively, the frequent loss of the p53 gene could be associated with the deletion of an adjacent gene, which could be involved in the pathogenesis of these diseases.     

p 53 gene rearrangements in chronic myelocytic leukemia

Summary We performed Southern-blot analysis of the p 53 gene in 41 consecutive patients with typical chronic myelocytic leukemia (CML). In two of them, we were able to study cells during both the chronic and the accelerated phases. Only one of the 29 chronic-phase samples had rearrangement of the p 53 gene, whereas three of the nine accelerated-phase samples and one of the five patients in blast crisis exhibited rearrangements. Gene deletion was observed in two patients, one in accelerated phase and the other in blast crisis. One patient with a nonrearranged p 53 gene in chronic phase showed rearrangement after progression to the accelerated phase. On the other hand, one patient in accelerated phase exhibited rearrangements which disappeared after reversion to chronic phase with successful treatment. Our findings support the opinion that alterations of the p 53 gene may play an important role in CML evolution.     

hnRNP B1特异性小干扰RNA对肺癌细胞及p53表达影响的研究进展

核内不均一核糖核蛋白(hnRNP)B1的高表达是肺癌频发早期事件,可能与细胞分化失调控有关. p53是目前公认的一种抑癌基因,当细胞受到各种信号刺激引起基因应激反应时,p53蛋白被磷酸化而变构从而被活化,抑制细胞的分裂、增殖和诱导细胞凋亡,防止细胞的恶性转化.应用RNA干扰技术中的小干扰RNA使hnRNP B1基因沉默,能使p53基因的活性增强,从而抑制细胞的恶性转化.本文就hnRNP B1特异性小干扰RNA对肺癌细胞p53基因表达影响的研究进展作一综述.     

白血病细胞p53和bcl-2表达与化疗药物诱导凋亡的关系

目的　探索p5 3和bcl 2表达及化疗药物体外诱导白血病细胞凋亡与患者化疗疗效的关系。方法　用免疫组化法测定 42例急性白血病患者骨髓细胞p5 3和bcl 2表达水平 ;用TdT介导的脱氧核苷酸切口和末端标记法 (Tunel法 )、Wright Giemsa染色及超微结构观察等方法检测化疗药物诱导的白血病细胞凋亡。结果　 42例急性白血病患者p5 3和bcl 2总体表达水平显著高于对照组。2 7例随访组患者完全缓解 (CR) 16例 ,未缓解 (NR) 11例。CR者表现以p5 3和bcl 2共同阴性及化疗诱导高凋亡为主 ,NR者以p5 3和bcl 2共同阳性及化疗诱导低凋亡占优势。形态学观察可见典型细胞凋亡特征。结论　检测急性白血病患者p5 3和bcl 2表达水平及体外化疗药物诱导白血病细胞凋亡的敏感性有助于预测患者的化疗效果。     

肿瘤抑制基因p53与肝炎病毒调节的研究

肝炎病毒包括甲、乙、丙、丁、戊型等肝炎病毒 ,而研究最多的是乙型肝炎病毒 (HBV)和丙型肝炎病毒(HCV)。p5 3是肿瘤抑制基因 ,编码p5 3蛋白直接诱导基因的表达并参与细胞周期的调节。肝炎病毒基因编码的蛋白也作用于肝细胞 ,引起肝脏的损害。肝炎病毒与p5 3互相作用 ,导致肝细胞病变 ,特别是肿瘤的发生。一、p5 3的结构和功能p5 3基因是肿瘤抑制基因 ,又称为基因组卫士 ,在DNA损害时 ,在修复过程中具有重要作用。在这种条件下 ,p5 3抑制细胞周期或诱导凋亡。p5 3蛋白的结构和功能分为 4个区 ,其中 2个区最重要 :序列特异DNA结合区、氨…     

The influence of the p53 gene on the in vitro chemosensitivity of colorectal cancer cells

Purpose : The p53 gene is considered one of the most important in the control of apoptosis, and its mutations have a close relationship with chemosensitivity. The aim of this work was to investigate the role of p53 in the apoptosis of colorectal cancer cells in vitro, induced by 5-fluorouracil (5-FU) and hydroxy-camptothecin (HCPT). Methods : A total of 39 colorectal cancer samples from patients were treated in vitro with 5-FU (10 μg/ml), 5-FU (10 μg/ml) + leucovorin (5 μg/ml), HCPT (0.1 μg/ml) and HCPT (0.1 μg/ml) + Salvia mitorrhiza (6 μl), using an in situ terminal deoxynucleotidyltransferase assay to detect chemosensitivity. p53 gene mutations from tumor DNA were detected, after amplification by the polymerase chain reaction of exons 5–8, by non-radioactive single-strand conformation polymorphism. Results : p53 gene mutations were observed in 43.6% (17/39) of colorectal carcinomas, when the terminal deoxynucleotidyltransferase assay was used to detect the tumor apoptotic rate. Cells with mutated p53 had lower chemosensitivity than those without (p < 0.01). Conclusion : Routine assessment of p53 status may be helpful in selecting patients with the wild-type p53 gene, who have a predictably better response to chemotherapy. Received: 18 November 1997 / Accepted: 17 December 1998     

人原发性肝癌细胞株受照射后凋亡与癌基因表达

目的 探讨人原发性肝癌细胞株受照射后凋亡与ｂｃｌ－２、ｐ５３基因表达产物关系。方法 选择人原发性肝癌细胞株ＱＧＹ,１８０ＫＶＸ线照射,流式细胞仪技术定量测定凋亡发生率,单克隆抗体免疫化法测定受照射后ｂｃｌ－２、ｐ５３表达产物,Ｓｏｎｙ Ｍｉａｓ－３００ Ｓｐａｔｏ图象分析仪分析结果。结果 ＱＧＹ－７７３０细胞株自发凋亡率为４．７９％,随照射后时间延长及照射剂量增加凋亡发生率增加。Ｂｃｌ－１、ｐ５３     

Early apoptosis and cell death induced by ATX-S10Na ( Ⅱ)-mediated photodynamic therapy are Bax- and p53-dependent in human colon cancer cells

AIM: To investigate the roles of Bax and p53 proteins in photosensitivity of human colon cancer cells by using lysosome-localizing photosensitizer, ATX-S10Na (Ⅱ).METHODS: HCT116 human colon cancer cells and Bax-null or p53-null isogenic derivatives were irradiated with a diode laser. Early apoptosis and cell death in response to photodynamic therapy were determined by MTT assays, annexin V assays, transmission electron microscopy assays, caspase assays and western blotting.RESULTS: Induction of early apoptosis and cell death was Bax- and p53-dependent. Bax and p53 were required for caspase-dependent apoptosis. The levels of anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-XL,were decreased in Bax- and p53-independent manner.CONCLUSION: Our results indicate that early apoptosis and cell death of human colon cancer cells induced by photodynamic therapy with lysosome-localizingphotosensitizer ATX-S10Na (Ⅱ) are mediated by p53-Bax network and Iow levels of Bcl-2 and Bcl-XL proteins.Our results might help in formulating new therapeutic approaches in photedynamic therapy.