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。     

p73基因与肿瘤

癌基因的激活和抑癌基因的失活是人类恶性肿瘤发生和发展的重要原因。p53基因是与人类肿瘤相关性最高的基因,近半数的恶性肿瘤与p53基因的突变有关,而p73基因是1997年发现的p53基因家族的一个成员,近年来对其结构、功能、分布和表达情况都有了深入的研究。我们就p73基因的研究现状以及其在人类肿瘤中的表达情况作一综述。p73基因定位于1p36.33,由13个外显子和12个内含子组成,表达相对分子质量为73×103的蛋白,在结构上与p53蛋白有同源性。p73过表达时能抑制细胞生长和诱导细胞凋亡,但是病毒原癌蛋白却与p73没有相互作用。C-Ab1激酶可以活化p73基因并参与凋亡反应。p73基因敲除的小鼠没有肿瘤易患现象发生,但却有严重的发育异常。p73基因在神经母细胞瘤中不常发生突变,在肺癌、食管癌、肝癌、卵巢癌、乳腺癌表达增高,在淋巴瘤和某些白血病表达降低。与p53基因有明显的不同,p73基因有自己独特的功能。对于野生型p73基因在多种人类肿瘤中过表达的一种解释是,p73基因的过表达可能是一种p53基因失活的代偿反应,虽然这种作用可能无法完全替代p53基因的作用。但是目前研究已经证实p73基因很少突变,而且在肿瘤组织中存在异常表达,增加其表达可能诱导肿瘤细胞的凋亡,并且在p53基因突变时起抑制肿瘤细胞增殖的作用。因此增强p73基因表达也许是恶性肿瘤治疗的一种新方法。     

78例大肠肿瘤中p53、FHIT的表达及临床意义

p53基因及脆性组氨酸三联体蛋白(fragile histidine triad FHIT)基因在大肠肿瘤“腺瘤—癌”的多阶段发生模式中起着重要作用^[1～2]。野生型p53蛋白是细胞增殖、分化及凋亡中的关键调节因子^[3]p53基因错义突变、产生变异蛋白、抑制细胞凋亡^[4]、促进细胞增殖，而发挥促癌作用。FHIT表达于大多数正常组织中。其基因结构及表达异常见于多种类型的肿瘤组织中^[5]。本文应用免疫组化方法，检测p53及FHIT在大肠肿瘤中的表达，以探讨p53及FHIT在大肠肿瘤所起的作用及临床意义。     

腺病毒介导p21WAF1/CIP1基因转移及诱导胃癌细胞凋亡

目的：探讨p21^WAF1/CIP1基因（p21基因）过表达对人胃癌细胞恶性表型和细胞凋亡的影响，进一步了解p21基因对肿瘤细胞的治疗作用。方法：通过腺病毒介导外源性p21基因转移到有p53基因突变的人胃癌细胞系SGC－7901，对p21基因的表达、细胞生长的抑制与投机和对肿瘤模型的治疗效果进行分析。结果：外源性p21基因在靶细胞高水平表达显著抑制了胃癌细胞的生长和集落形成，流式细胞仪检测显示G1期阻滞并发生了凋亡。瘤内注射Ad-p21对裸鼠体内移植瘤有一定抑瘤作用。结论；p21基因可能参与肿瘤细胞凋亡的诱导，使p21基因在肿瘤的基因治疗，特别是在与其他凋亡诱导因子联合作用的方案中有更好的应用前景。     

miR-409-3p通过抑制LHX2调控VEGF/VEGFR2信号通路促进前列腺癌细胞凋亡、抑制细胞增殖

目的:探讨微小RNA-409-3p（microRNA-409-3p,miR-409-3p）对前列腺癌细胞增殖、凋亡的影响及其分子机制。方法:qRT-PCR与Western blot平行检测前列腺癌细胞中miR-409-3p与LIM同源框基因2（LIM-homeobox gene 2,LHX2）的表达;CCK-8实验检测miR-409-3p过表达对前列腺癌细胞增殖能力的影响,以及LHX2过表达对前列腺癌细胞增殖能力的恢复作用;流式细胞术检测细胞凋亡率;双荧光素酶报告基因检测miR-409-3p与LHX2的靶向作用关系;Western blot检测细胞中LHX2、CyclinD1、CDK4、Cleaved-caspase-3及VEGF/VEGFR2信号通路相关蛋白表达。结果:miR-409-3p在前列腺癌细胞中的表达水平显著低于正常前列腺上皮细胞（P＜0.05）,LHX2的表达水平高于正常前列腺上皮细胞（P＜0.05）;miR-409-3p过表达可显著抑制前列腺癌细胞增殖,促进细胞凋亡（P＜0.05）,并可上调Cleaved-caspase-3的表达（P＜0.05）,下调CyclinD1、CDK4、VEGF、t-VEGFR2、p-VEGFR2的表达（P＜0.05）;双荧光素酶实验证明miR-409-3p与LHX2存在靶向关系,miR-409-3p可负性调控LHX2的表达;LHX2过表达可部分逆转miR-409-3p对前列腺癌细胞增殖、凋亡及VEGF/VEGFR2信号通路的影响。结论:miR-409-3p可通过下调LHX2的表达而促进前列腺癌细胞凋亡、抑制细胞增殖,其作用机制可能与抑制VEGF/VEGFR2信号通路激活有关。     

增殖细胞核抗原和p27在前列腺增生和前列腺癌组织中的表达及意义

目的　探讨增殖细胞核抗原 (PCNA)和p2 7蛋白在前列腺增生及前列腺癌组织中的表达 ,及其与肿瘤发生发展的关系。方法　应用免疫组化S P法 ,检测 30例前列腺增生和 37例前列腺癌石蜡包埋存档标本组织中PCNA、p2 7蛋白的表达 ,对其在前列腺增生及前列腺癌不同病理分级、临床分期中的表达进行对比分析和相关性研究。结果　前列腺增生组织的PCNA强阳性表达率(3.3% )与前列腺癌组织 (83.8% )相比 ,差异有显著性 (P <0 .0 1)。前列腺增生组织中p2 7蛋白高表达 ( )率 (70 .0 % )与前列腺癌 (2 7.0 % )相比 ,差异有显著性 (P <0 .0 5 ) ,p2 7蛋白表达与前列腺癌组织的分化程度及临床分期无关 (P >0 .0 5 )。前列腺增生组织中 ,PCNA与p2 7蛋白表达呈负相关 (P <0 .0 1) ;前列腺癌组织中 ,PCNA与p2 7蛋白表达无相关性 (P >0 .0 5 )。结论　p2 7蛋白表达减少或缺失与前列腺肿瘤的发生有关 ,而与恶性肿瘤的进展无关 ,其表达的缺失是前列腺癌重要的阴性预测因子 ;PCNA表达程度不仅与前列腺增生及前列腺癌的发生有关 ,而且与前列腺癌的恶性行为有关 ,PCNA的检测对判断肿瘤的性质、转移及预后有重要的临床意义     

mdm2与p53基因相互作用分子机理研究新进展

mdm2基因是与人类肿瘤密切相关的癌基因,它与p53基因间存在相互调节关系,p53能刺激mdm2基因的表达,mdm2’基因可以促进p53蛋白降解、抑制p53基因介导的反式激活和细胞凋亡功能.mdm2对p53基因功能的负性调节在部分含有野生型p53基因肿瘤的发生、发展中可能起着重要的作用,阻断两者间的相互作用以恢复p53基因的抑癌功能为恶性肿瘤基因治疗的研究提供了新思路.本文综述两者相互作用分子机理研究的最新进展.     

靶向沉默核干因子对前列腺癌PC-3细胞全基因组表达谱变化的影响

目的 探讨核干因子(NS)基因在前列腺癌恶性增殖中的作用机制.方法 采用表达谱基因芯片技术,分析前列腺癌PC-3细胞NS基因表达下调后全基因组表达谱的变化,筛选与NS相互作用的差异表达基因及信号通路.采用实时荧光定量聚合酶链反应对重要的差异表达基因进行验证.结果 共筛选出219个差异表达的基因,这些基因涉及到细胞周期、细胞增殖、信号转导、细胞凋亡和细胞分化等多个方面.NS基因表达下调后主要引起周期素依赖激酶4抑制因子(INK4)家族基因(p15、p16和p18)的上调,以及cyclin D1和HDACI基因的下调,其主要作用点位于CDK4/6-cyclinD和pRb-E2F1复合体上.结论 在前列腺癌PC-3细胞中,NS基因可能主要通过抑制p15、p16和p18等INK4家族的肿瘤抑制基因的表达,从而促进肿瘤的发生和发展;NS基因是细胞周期G1/S期检查点的重要调控因子.     

细胞周期蛋白依赖性激酶抑制剂与肿瘤相关性的研究进展

细胞周期蛋白依赖性激酶抑制剂相关基因的突变和表达失调可直接或间接影响细胞的周期、增殖以及凋亡等功能,与肿瘤的发生发展密切相关。p16能够抑制CDK4／CDK6介导的Rb蛋白产物的磷酸化,其CpG岛异常甲基化参与宫颈癌的发生发展;p21作为p53的下游调节因子,与肿瘤血管的生成、淋巴转移及预后相关;p27作用机制复杂,与乳腺癌预后关系密切,并可恢复耐药乳腺癌细胞对他莫昔芬的敏感性;p57在细胞周期G1到S期的转变中至关重要,其表达程度与部分肿瘤的恶性程度相关,它还参与了肿瘤细胞的凋亡过程。了解这些基因的结构和作用机制,探究其在肿瘤发生、发展中所产生的作用,进而寻找有效治疗靶点,已成为肿瘤分子生物学关注的热点。     

p73基因与消化系统肿瘤

p73基因是肿瘤抑制基因p53家族的新成员,在编码蛋白的结构、功能等方面两者很相似,但在其它方面有非常显著的差异。许多研究发现p73基因异常与神经母细胞瘤、黑色素瘤、前列腺癌、肺癌有关。近年来还发现p73基因可能参与了一部分消化系统肿瘤的发生发展。本文综述了p73基因的研究现状及其与消化系统肿瘤的发生、发展、预后的关系及表达情况,从而认为,对p73基因的深入研究将有助于人们进一步认识肿瘤的本质,为肿瘤基因治疗提供新的靶点。     

Biological factors and therapeutic modulation in prostate cancer radiotherapy

Biologic factors affect the ability of radiation to effectively treat all patients with prostate cancer. The use of prognostic and genetic markers (12 lipoxygenase, p53, bc1-2 genes, ploidy) may aid in the development of treatments for these patients. Particularly, several studies have shown that p53 tumor suppressor gene mutations are infrequent in prostate cancer and are associated with advanced disease. Recent efforts have been directed toward novel therapeutic modalities, especially in combination with standard irradiation of prostate carcinoma. These modalities are based on an improved knowledge of these biological factors involved in the progression and diffusion of the tumor, especially p53. Several authors evaluated the predictive and prognostic role of p53 in patients with prostatic cancer treated with radiotherapy. An extensive review of international reports on the subject is provided.     

Androgen receptor expression in relation to apoptosis and the expression of cell cycle related proteins in prostate cancer

The expression of several genes involved in the regulation of cell cycle and apoptosis may be regulated via the androgen receptor (AR) in the prostate. AR may have a role in the prognosis of prostatic carcinoma. The aim was to examine AR expression status and its relationship with markers of proliferation, apoptosis and cell cycle control in prostate cancer. Expression of AR, bcl-2, bax, Ki-67 and p53 was examined in paraffin-embedded tissues from 50 cases of prostate carcinoma by immunohistochemistry and evaluated using an index of staining. Detection of apoptotic cells was performed by TUNEL method. Correlation between AR expression and apoptosis, proliferation index, bcl-2, bax and p53 and also clinicopathological parameters including stage, pathological grade and Gleason score were determined. AR expression was observed in all cases with mean expression of 81%±15 and mean staining index of 141±65. No correlation was found between AR expression and apoptosis detected in patients. The mean AR staining index was 170±72 in bcl-2 positive tumors versus 120±53 in bcl-2 negative tumors showing a significant association between AR and bcl-2 expression (p=0.015). AR expression also showed a significant association with bcl-2/bax ratio (r=0.321, p=0.023) and Ki-67 proliferation staining index (r=0.396, p=0.004). Although a significant correlation between Ki-67 and p53 with differentiation status of the tumors was observed (p<0.004) no correlation was found with AR. AR expression showed no prognostic value regarding its correlation with stage and differentiation status of the prostate carcinoma. However, its significant correlation with Ki-67 and bcl-2 that are markers of cell survival suggest its contribution to tumor cell progression.     

p53 Arg72Pro polymorphism predicts survival outcome in lung cancer patients in Indian population

In response to many forms of cellular stress, including DNA damage, the p53 protein functions to induce growth arrest, DNA repair, or apoptosis. Common allele variants in the TP53 gene modulate pathways of lung carcinogenesis and susceptibility to or prognosis of lung cancer. The prognostic role of the polymorphism was assessed in 422 subjects using PCR-RFLP. Logistic regression analysis showed a dominant presentation of Pro/Pro homozygotes in lung carcinoma population than in control population (OR = 2.1, P = 0.003). We further investigated the association of p53 codon 72 polymorphism with prognosis in 170 lung cancer patients. Kaplan-Meier survival analyses showed a significant difference in survival between p53 variant genotypes and overall survival (P = 0.02). Cox regression analysis showed p53 Arg72Pro heterozygous genotype was overall an independent prognostic factor (Risk ratio of death, 2.2; P = 0.02), suggesting Pro72Pro genotype to be a potential risk factor favoring the development of lung carcinoma and that Arg72Pro genotype is independently associated with a poorer prognosis of lung cancer.     

E2F1在肿瘤中的研究进展

E2F1是E2F转录因子家族的重要一员，参与人体生命活动中重要的细胞周期和细胞凋亡环节，E2F1促进S期的细胞增多，同时通过p53依赖和非依赖途径诱导细胞凋亡。近来研究显示，E2F1在多种肿瘤如肺癌、前列腺癌、乳腺癌等组织细胞中呈高表达，表现为促癌基因的作用，其表达水平与肿瘤的发生、发展及预后密切相关。本文结合国内外最新研究报道，对E2F1在肿瘤中的研究进展作一综述。     

Antiproliferative and proapoptotic activities of triptolide (PG490), a natural product entering clinical trials, on primary cultures of human prostatic epithelial cells.

Interest in exploiting traditional medicines for prevention or treatment of cancer is increasing. Extracts from the herb Tripterygium wilfordii hook F have been used in China for centuries to treat immune-related disorders. Recently it was reported that triptolide (PG490), a purified compound from Tripterygium, possessed antitumor properties and induced apoptosis by p53-independent mechanisms in a variety of malignant cell lines. This property of triptolide attracted our attention because we have found that primary cultures of human prostatic epithelial cells derived from normal tissues and adenocarcinomas are in general extremely resistant to apoptosis. Furthermore, the function of wild-type p53 is impaired in these cells such that drugs that require p53 activity to induce cell death are ineffective. Therefore, the properties of triptolide and the recent approval of its water-soluble form (PG490-88) for entry into Phase I clinical trials suggested that this drug was a promising candidate to test for antitumor activity against prostate cells. Experiments presented here demonstrated that triptolide had dose-dependent effects on both normal and cancer-derived primary cultures of human prostatic epithelial cells. Low concentrations of triptolide inhibited cell proliferation and induced a senescence-like phenotype. Higher concentrations of triptolide induced apoptosis that was unexpectedly associated with nuclear accumulation of p53. Paradoxically, levels of the p53 target genes, p21(WAF1/CIP1) and hdm-2, were reduced, as was bcl-2. Our preclinical studies suggest that triptolide might be an effective preventive as well as therapeutic agent against prostate cancer and that triptolide may activate a functional p53 pathway in prostate cells.     

Prognostic value of immunohistochemical expression of the c-erbB-2 oncoprotein in metastasic prostate cancer.

It is well established that the activation of proto-oncogenes could trigger uncontrolled cell growth and cancer development. Although this correlation has already been evidenced in several human tumors, no conclusive studies have related oncogene activation with the development of prostatic neoplasia. Nevertheless, some reports suggest that c-erbB-2, which is a prognostic marker in breast cancer, could be implicated in the development of prostatic adenocarcinoma. We have studied the expression of the c-erbB-2 oncoprotein in primary prostatic tissue in a series of 70 patients with metastasic disease, by means of immunohistochemistry. The NCL-B 11 anti-c-erbB-2 monoclonal antibody was used, and the immunoreactivity was quantified by image analysis. The overall rate of prostatic-tissue sections presenting positive c-erbB-2 immunostaining was 64.3%. No significant relation was observed between histological grade and c-erbB-2 over-expression or severity of the disease, based on the extent of metastases. The average specific survival in patients with c-erbB-2 over-expression was 33 months, while it was 54 in patients with c-erbB-2 negativity; p < 0. 034. These results, as well as the logistic-regression analysis, suggest that expression of c-erbB-2 oncoprotein would be considered as an independent prognostic factor of metastatic prostate cancer. Moreover, it could discriminate between the prognosis of patients with Gleason score 2 to 7 and those with score 8 to 10. Our results suggest that the expression of c-erbB-2 oncoprotein in primary prostatic tissue could have a prognostic value in patients with metastatic prostate cancer. Int. J. Cancer (Pred. Oncol.) 84:421-425, 1999.     

Regulation of Akt/PKB activity, cellular growth, and apoptosis in prostate carcinoma cells by MMAC/PTEN.

Understanding the functional roles of the molecular alterations that are involved in the oncogenesis of prostate cancer, the second most frequent cause of cancer-related deaths among men in the United States is the focus of numerous investigations. To examine the possible significance of alterations associated with the tumor suppressor gene, MMAC/PTEN, in prostate carcinoma, the biological and biochemical effects of MMAC/PTEN expression were examined in LNCaP cells, which are devoid of a functional gene product. Acute expression of MMAC/PTEN via an adenoviral construct resulted in a dose-dependent and specific inhibition of Akt/PKB activation, consistent with the phosphatidylinositol phosphatase activity of MMAC/PTEN. MMAC/PTEN expression induced apoptosis in LNCaP cells, although to a lesser extent than that observed with p53 via an adenoviral construct. However, MMAC/PTEN expression produced a growth inhibition that was significantly greater than that achieved with p53. Overexpression of Bcl-2 in LNCaP cells blocked MMAC/PTEN- and p53-induced apoptosis but not the growth-suppressive effects of MMAC/ PTEN, suggesting that the growth regulatory effects of MMAC/PTEN involve multiple pathways. These studies further implicate the loss of MMAC/PTEN as a significant event in prostate cancer and suggest that reintroduction of MMAC/PTEN into deficient prostate cancer cells may have therapeutic implications.     

Molecular markers of prostate cancer outcome

Molecular markers have the potential to serve not only as prognostic factors but may be targets for new therapeutic strategies and predictors of response in a range of cancers. Prostate cancer development and progression is predicated on a series of genetic and epigenetic events within the prostate cell and its milieu. Within this review, we identify candidate molecules involved in diverse processes such as cell proliferation, death and apoptosis, signal transduction, androgen receptor (AR) signalling, cellular adhesion and angiogenesis that are linked to outcome in prostate cancer. Current markers with potential prognostic value include p53, Bcl-2, p16INK4A, p27Kip1, c-Myc, AR, E-cadherin and vascular endothelial growth factor. Evolving technology permits the identification of an increasing number of molecular markers with prognosis and predictive potential. We also review the use of gene microarray analysis in gene discovery as a means of identifying and cosegregating novel markers of prostate cancer outcome. By integrating selected markers into prospective clinical trials, there is potential for us to provide specific targeted therapy tailored for an increasing number of patients.     

Limitations of tissue microarrays in the evaluation of focal alterations of bcl-2 and p53 in whole mount derived prostate tissues

Several investigators have reported the correlation of p53 and bcl-2 immunoreactivity with post operative prostate specific antigen (PSA) recurrence. Focal and or clustered expression is typical for these biomarkers. The purpose of this study was to compare the effectiveness of tissue microarrays to detect p53 and bcl-2 overexpression and their prognostic significance. Tissue microarrays (TMA) of 99 patients with mean follow-up of 61 months contained 760 samples from 241 carcinomas, 431 benign glands, and 88 foci of prostatic intraepithelial neoplasia (PIN). Overexpression of p53 was seen in 43.3% of 97 patients, whereas bcl-2 overexpression was noted in 23.7% of 97 patients using TMA technology, compared to 66.0% and 26.9%, respectively in the corresponding radical prostatectomy samples. The tissue microarray technology is a powerful tool to study the multifocal and heterogeneous nature of prostate cancer. However, the prognostic value of p53 and bcl-2 could not be confirmed using this technology in contrast to radical prostatectomy sections. The TMA technique is probably more informative and reliable in evaluating the prognostic value of homogeneously expressed biomarkers.