p21 WAF1/CIP1基因导入对p53突变人肺癌细胞系生长特性的影响

目的　探讨含突变ｐ５３ 基因癌细胞系表达ｐ２１ 基因的抗肿瘤作用。方法　构建人ｐ２１表达重组子,导入ｐ５３ 基因突变的人肺癌ＰＧ细胞系,建立稳定高表达ｐ２１ 的细胞系;然后观察其形态及生长特性的改变;ＣＤＫ４ ,ＰＣＮＡ水平;对基因毒性物的反应性。结果　在含ｐ５３ 基因突变的ＰＧ 细胞内导入外源性ｐ２１ 基因,其高表达ｐ２１ 约是ＰＧ 细胞的６ 倍;转染细胞出现核规则、核膜变薄等异型性降低;生长速度下降（５０％ ） ,叠落生长不明显;在０ ．５％ 血清条件下生长显著低于对照组;血清依赖性升高;软琼脂集落实验：转染细胞集落形成率降低为１ ％ （ 对照组为２８％ ） 。ｗｅｓｔｅｒｎ ｂｌｏｔ 显示,ｐ２１ 转染细胞的ＣＤＫ４ 和核仁组成区相关蛋白水平显著降低。但顺铂诱导下转染细胞凋亡发生延缓至４８ 小时以后（ 对照组２４ 小时）。结论　在有ｐ５３ 基因突变的肿瘤细胞ｐ２１ 的高表达仍能抑制癌细胞的生长,降低恶性表型;其作用可能与降低细胞ＣＤＫ４ 和ＰＣＮＡ 水平有关,而不依赖细胞凋亡机制。表达ｐ２１ 基因可作为恢复ｐ５３ 基因功能的旁路途径。     

野生型p53基因诱导肝癌细胞凋亡及p21（WAF1／CIP …

目的：研究野生型ｐ５３基因对人肝癌细胞系细胞凋亡的作用。方法：通过脂质体转染方法将野生型ｐ５３基因（ｗｔ－ｐ５３）导入ｐ５３缺陷的ＨＣＣ－９２０４细胞系中，并获稳定表达。结果：转染ｗｔ－ｐ５３后细胞生长缓慢，Ｇ１期细胞数量由转基因前的４８．５％增加到７８．０％，并有较多细胞逐渐死亡。电镜观察和ＤＮＡ分析证实细胞死亡方式主要是细胞凋亡。免疫组化结果显示细胞转染ｗｔ－ｐ５３后，ｐ２１ＷＡＦ１／ＣＩＰ１     

野生型p53基因诱导肝癌细胞凋亡及p21(WAF1/CIP1)蛋白过表达

目的：研究野生型ｐ５３基因对人肝癌细胞系细胞凋亡的作用。方法：通过脂质体转染方法将野生型ｐ５３基因（ｗｔ－ｐ５３）导入ｐ５３缺陷的ＨＣＣ－９２０４细胞系中，并获稳定表达。结果：转染ｗｔ－ｐ５３后细胞生长缓慢，Ｇ１期细胞数量由转基因前的４８．５％增加到７８．０％，并有较多细胞逐渐死亡。电镜观察和ＤＮＡ分析证实细胞死亡方式主要是细胞凋亡。免疫组化结果显示细胞转染ｗｔ－ｐ５３后，ｐ２１ＷＡＦ１／ＣＩＰ１的表达显著增加。结论：提示外源性野生型ｐ５３基因可以抑制肝癌细胞生长，并诱导细胞凋亡，这可能是通过ｐ２１ＷＡＦ１／ＣＩＰ１途径发挥上述作用的     

脂质体介导的p53基因对肝癌细胞生长的抑制作用

目的 观察外源野生型ｐ５３基因在肝癌基因治疗方面的可行性。方法 将载有人野生型ｐ５３－ｃＤＮＡ的真核表达质粒ｐ５３－ｐｃＤＮＡ３,用阳离子脂质体介导转染人肝癌细胞系ＨｅｐＧ２,用流式细胞仪检测ｐ５３－ｐｃＤＮＡ３对ＨｅｐＧ２细胞生长的影响。结果 通过观察细胞生长曲线与流式细胞仪检测细胞周期和细胞的凋亡指数发现,ＨｅｐＧ２细胞生长受到明显的抑制。结论 脂质体介导的ｐ５３基因可在Ｈ细胞中表达,且明显抑     

膀胱移行细胞癌P21,P185,p53蛋白表达及ras,p53基因突变的检测

为探讨基因的改变在膀胱移行细胞癌发生过程中的意义，应用免疫组织化学及聚合酶链反应－限制性片段长度多肽性法，检测１５０例膀胱移行细胞癌Ｐ２１、Ｐ１８５、ｐ５３蛋白表达以及５０例ｒａｓ、ｐ５３基因突变。结果表明：膀胱移行细胞癌Ｐ２１、Ｐ１８５、ｐ５３蛋白表达阳性率分别为５９．３％（８９例）、５５．３％（８３例）、２９．３％（４４例）。Ｐ２１、ｐ１８５蛋白表达阳性率与膀胱移行细胞癌分级呈负相关（Ｐ＜０．０５），ｐ５３蛋白表达与膀胱移行细胞癌分级呈正相关（Ｐ＜０．０１）。Ｐ２１、ｐ５３蛋白表达阳性率与预后呈显著相关，Ｐ２１与预后呈负相关。７３例膀胱移行细胞癌存在两种以上蛋白共同表达。膀胱移行细胞癌Ｈａ－ｒａｓ癌基因第１２位密码子突变１６例（３２％），ｐ５３基因突变９例（１８％），均为２４８位点突变。Ｈａ－ｒａｓ第１２位点突变随病理分级增高而增高，与膀胱移行细胞癌分级显著相关（Ｐ＜０．０５）。ｒａｓ基因、ｐ５３基因突变与预后显著相关（Ｐ＜０．０５）。ｒａｓ基因、ｐ５３基因突变患者死亡率高于无突变患者。４例膀胱移行细胞癌存在ｒａｓ基因及ｐ５３基因共同突变。     

外源性p53基因对人胃粘膜上皮细胞系GES－1的生物学效应

为了解ｐ５３基因在细胞癌变过程中的作用，分别将野生型和突变型的ｐ５３基因导入体外培养的人胃粘膜上皮细胞系ＧＥＳ－１中，进一步比较，分析转染ｐ５３基因对ＧＥＳ－１细胞的生物学效应发现：（１）外源ｐ５３基因改变了ＧＥＳ－１细胞的形态──转染野生型ｐ５３基因使得ＧＥＳ－１出现一些细胞分化的迹象，在其胞浆有明显的空泡，空泡周围有微管的环绕；（２）ｐ５３基因的转染改变了ＧＥＳ－１生长状态──野生型的ｐ５３基因转染的细胞的克隆形成率低于突变型ｐ５３基因以及空载体ｎｅｏ转染的细胞。实验还发现突变型ｐ５３基因改变了细胞生长习性，使细胞有选择生长优势，对细胞恶性度有促进作用。     

外源性p53基因对人胃粘膜上皮细胞系GES—1的生物学效应

为了解ｐ５３基因在细胞癌变过程中的作用，分别将野生型和突变型的ｐ５３基因导入体外培养的人胃粘膜上皮细胞系ＧＥＳ－１中，进一步比较，分析转染ｐ５３基因对ＧＥＳ－１细胞的生物学效应发现：（１）外源ｐ５３基因改变了ＧＥＳ－１细胞的形态－转染野生型ｐ５３基因使得ＧＥＳ－１出现一些细胞分化的迹象，在其胞浆有明显的空泡，空泡周围有微管的环绕；（２）ｐ５３基因的转总收入籽ＧＥＳ－１生长状态－野生型的ｐ５３基因转     

一种特异点突变型p53minigene四环素负调控表达模式的抑瘤效应

目的建立四环素负调控的真核表达调控模式,进一步证实一种特异点突变型小鼠ｐ５３ｍｉｎｉｇｅｎｅ（密码子１７２Ａｒｇ→Ｌｅｕ）的强抑瘤效应。方法在观察基因转染瞬时表达凋亡效应和抗性克隆的形成能力的基础上,进一步应用基因重组方法,构建四环素负调控型的特异点突变型小鼠ｐ５３ｍｉｎｉｇｅｎｅ的真核表达载体,通过ＬｉｐｏｆｅｃｔａＭＩＮＥ基因转染法,导入ｐ５３杂合性缺失的人肺癌细胞ＰＧ,Ｐｕｒｏｍｙｃｉｎ筛选基因转染的稳定克隆,动态观察四环素调控表达及其对体外培养的人肺癌细胞ＰＧ的调控抑瘤效应。结果特异点突变型小鼠ｐ５３ｍｉｎｉｇｅｎｅ具有很强的体外抑瘤效应,瞬时表达能导致ＰＧ细胞凋亡,强于野生型（Ｐ＜０．０５）,并能抑制Ｇ４１８抗性克隆的形成;成功地建立了四环素负调控的特异点突变型ｐ５３ｍｉｎｉｇｅｎｅ真核表达调控转基因细胞模型,去除四环素和传代能导致细胞凋亡效应。结论特异点突变型ｐ５３ｍｉｎｉｇｅｎｅ具有恶性肿瘤基因治疗的应用价值,四环素负调控启动子元件能对基因进行真核表达调控,对基因治疗研究具有重要意义。     

转染野生型p53基因稳定表达后早期肝癌细胞的自发性凋亡

目的：研究外源性野生型ｐ５３基因（ｗｔ－ｐ５３）对ＨＣＣ－９２０４肝癌细胞系的作用和意义。方法：通过脂质体介导方式将人ｗｔ－ｐ５３基因转染至肝癌细胞中。结果：转染ｗｔ－ｐ５３稳定表达早期，肝癌细胞发生自发凋亡，流式细胞仪检测结果显示典型的凋亡峰，其凋亡细胞数为５２．８％，细胞周期分析结果显示，转染ｗｔ－ｐ５３细胞中Ｇ１、Ｓ和Ｇ２期细胞分别为７９．１％，２０．５％和０．４％，亲本细胞则分别为４８．５％，２７．０％和２３．７％。琼脂糖电泳显示ＤＮＡ断裂呈梯状，透射电镜观察肝癌细胞发生染色质浓聚并边集。结论：转染的外源性ｗｔ－ｐ５３具有介导肝癌细胞凋亡的作用。     

外源性野生型p53基因对人肺腺癌细胞系GLC-82的生物学效应

目的：研究外源性野生型ｐ５３基因对人肺腺癌细胞系ＧＬＣ－８２的生物学作用。方法：将含有野生型ｐ５３基因的ｐＤＯＲ－ｎｅｏ逆转录病毒载体，通过ｌｉｐｏｆｅｃｔｉｎ转染ＧＬＣ－８２细胞，用流式细胞仪、电镜及３Ｈ－ＴｄＲ掺入法，观察野生型ｐ５３基因对ＧＬＣ－８２细胞的生物学效应。结果：电镜观察可见，转染野生型ｐ５３基因，可使ＧＬＣ－８２细胞出现一些病理现象，如胞浆中有明显的空泡及线粒体肿胀；流式细胞仪分析显示，野生型ｐ５３基因可阻止ＧＬＣ－８２细胞周期停止于Ｇ１～Ｓ区；３Ｈ－ＴｄＲ掺入试验提示，野生型ｐ５３基因能够抑制ＧＬＣ－８２细胞ＤＮＡ的合成。结论：这些结果阐明了野生型ｐ５３基因是抑制ＧＬＣ－８２细胞生长的部分机理。     

Prognostic significance of p21WAF1/CIP1, p27Kip1, p53 and E-cadherin expression in gastric cancer

BACKGROUND: Gastric carcinoma is characterised by numerous genetic and epigenetic alterations that influence cell cycle progression, apoptosis and DNA repair. These alterations include down-regulation of the cyclin-dependent kinase (CDK) inhibitors p21(WAF1/CIP1) and p27(Kip1), and mutations of the tumour suppressor protein p53 and the cell adhesion molecule E-cadherin. Combined evaluation of the prognostic significance of these alterations has not been reported in Mexican Mestizo patients. AIMS: To evaluate p21(WAF1/CIP1), p27(Kip1), p53 and E-cadherin protein expression, including mutant E-cadherin variants with deletion of exon 8 (del 8) or 9 (del 9), in gastric cancer from Mexican patients. METHODS: Immunohistochemistry for the above-mentioned markers, including mutation-specific E-cadherin antibodies, was carried out in 69 gastric carcinomas; expression levels were correlated with histotype, tumour stage and prognosis. RESULTS: Expression of p21(WAF1/CIP1) alone or in combination with p27(Kip1) or in the absence of p53 was associated with favourable prognosis. Staining of del 8 and del 9 E-cadherin was found exclusively in patients negative for p53 and positive for p21(WAF1/CIP1), suggesting that the p21(WAF1/CIP1) regulatory function of p53 was intact. CONCLUSION: Combined evaluation of the prognostic significance of cell cycle regulators and E-cadherin should be performed. Even though patients negative for p53 and positive for p21(WAF1/CIP1) have a favourable prognosis, it may have a negative influence on prognosis if they acquire in addition E-cadherin mutations which have been shown previously to be associated with poor survival.     

Expression of p21 WAF1/CIP1 protein in colorectal cancers: study of its relation to p53 mutation and Ki67 antigen expression

Mutations of the p53 gene are the most common genetic alteration in malignant human tumors. A cyclin-dependent kinase inhibitor, p21WAF1/CIP1, is thought to be an important mediator of p53-induced cell cycle arrest. Although numerous studies have reported p53 expression and mutation in colorectal cancer few of them have correlated p53 expression with that of its downstream effector p21 and with the proliferation index as measured by expression of the Ki67 nuclear antigen. We studied p53, p21 and Ki67 expression by immunohistochemistry and molecular biology in 35 colorectal carcinomas. We compared these findings with each other and with clinical factors. Sixty three percent of tumors expressed p53 whereas seventy one percent expressed p21WAF1/CIP1. In adenocarcinomas, p21 staining was heterogeneous: p21-reactive cells were seen in the most differentiated areas. There was no correlation between p21WAF1/CIP1 and p53 expression, p53 mutation, Ki67 expression or clinical factors such as sex or location of the tumor. On the other hand, there was a statistical relationship between p21 expression and survival: our results indicated an association between high p21 expression and lower stages p21WAF1/CIP1 appears to be induced independently of p53 in these tumors and may be associated with differentiation rather than proliferation.     

p21WAF1/CIP1 expression in colorectal carcinoma correlates with advanced disease stage and p53 mutations

Defects in the mechanisms controlling the cell cycle are crucial in cell transformation and/or tumour progression. p21^WAF1/CIP1 is an inhibitor of cyclin-dependent kinases, induced by p53-dependent and p53-independent pathways, which can block progression through the cell cycle. p21^WAF1/CIP1 expression has been investigated immunohistochemically in a series of 191 patients with colorectal cancer of known p53 status. The purpose of the study was two-fold: to assess the relationship between p21^WAF1/CIP1 immunoreactivity and p53 alterations, and to evaluate the prognostic significance of p21^WAF1/CIP1 expression. In 96 carcinomas (51 per cent), p21^WAF1/CIP1 was expressed in over 10 per cent of tumour cells, whereas in 26, p21^WAF1/CIP1 was detected in under 10 per cent of neoplastic cells; 69 tumours lacked p21^WAF1/CIP1 expression. Immunoreactivity was more frequent in tumours of the right colon (p < 0·003) and was inversely correlated with tumour stage (p < 0·03), p53 gene mutations (p < 0·0007), p53 protein accumulation (p < 0·019), and Bcl-2 expression (p < 0·0005). In univariate analysis, down-regulation of p21^WAF1/CIP1 expression was associated with poor overall (p = 0·0022) and disease-free survival (p = 0·0009). Multivariate analysis, however, did not confirm any independent prognostic significance of p21^WAF1/CIP1 expression. The results indicate that p21^WAF1/CIP1 is associated with abnormal accumulation of p53 protein and the occurrence of p53 gene mutations in colorectal cancer and that lack of p21^WAF1/CIP1 expression is correlated with reduced patient survival in univariate analysis. These data underline the crucial pathogenetic role of the p53–p21^WAF1/CIP1 pathway in carcinomas of the large bowel. Copyright © 1999 John Wiley & Sons, Ltd.     

p21WAF1/CIP1 expression in gestational trophoblastic disease: correlation with clinicopathological parameters, and Ki67 and p53 gene expression.

BACKGROUND: The p21WAF1/CIP1 gene mediates growth arrest by inhibiting G1 cyclin dependent kinases and has been considered as a downstream effector of the tumour suppressor gene p53. AIM: To analyse the role of p21WAF1/CIP1 in gestational trophoblastic disease. METHODS: The immunohistochemical expression of p21WAF1/CIP1 gene was measured in 33 placentas, 28 partial hydatidiform moles, 54 complete hydatidiform moles, and 13 choriocarcinomas in paraffin wax embedded tissue. The results were correlated with p53 (DO7) and Ki67 (MIB1) immunoreactivity as well as clinical progress. RESULTS: p21WAF1/CIP1 immunoreactivity was found predominantly in the nuclei of the syncytiotrophoblasts. p21WAF1/CIP1 protein expression correlated with gestational age in normal placentas (p = 0.0001) but not in hydatidiform moles (p = 0.89). Complete hydatidiform moles and choriocarcinomas had a significantly higher p21WAF1/CIP1 expression compared with normal placentas and partial hydatidiform moles (p < 0.001); there was no difference between placentas and partial hydatidiform moles. No correlation between p21WAF1/CIP1 expression and either the proliferation (Ki67) index (p = 0.34) or p53 protein accumulation (p = 0.68) was demonstrated. There was no significant difference (p > 0.05) in p21WAF1/CIP1 expression between the 17 patients who developed persistent gestational trophoblastic disease and those who did not. CONCLUSIONS: This study suggests that p21WAF1/CIP1 expression in trophoblastic disease may be induced by a p53 independent pathway. The proliferative activity of gestational trophoblastic diseases might not be determined solely by the control of the cell cycle operated by p21WAF1/CIP1. p21WAF1/CIP1 expression is not an accurate prognostic indicator of gestational trophoblastic disease.     

膀胱移行细胞癌细胞周期调节相关基因表达与肿瘤生物学行为的关系

目的：探讨细胞周期调节相关基因p16INK4,p21WAF／CIP1,p53mlt在膀胱移行细胞癌中的表达与肿瘤增殖能力,病理分级及临床分期的关系。方法：应用免疫组织化学技术分析77例膀胱移行细胞癌组织p16INK4,p21WAF/CIP1,p53mlt基因的表达和增殖细胞核抗原（PCNA）表达情况,并与病理分级及临床分期之间进行综合分析。结果：p16INK4,p21WAF/CIP1,p53mlt在膀胱移行细胞癌中的表达及PCNA增殖指数与肿瘤的病理分级有关,与临床分期无关。p16,p21,p53阳性组与阴性组分别比较,其PCNA增殖数之间有差异性。多因素分析发现,p16INK4和p21WAF／CIP1阴性及p53mlt阳性组的PCNA值明显高于p16INK4和p21WAF／CIP1阳性及p53mlt阳性组,两者比较不同病理分级的阳性表达构成比亦有显著性差异。结论:联合检测p16INK4,p21WAF/CIP1,p53mlt基因的表达情况能充分反映膀胱移行细胞癌的增殖能力及生物学行为,对膀胱移行细胞癌患者的预后判断及治疗有指导意义。     

Oxygen induces S-phase growth arrest and increases p53 and p21(WAF1/CIP1) expression in human bronchial smooth-muscle cells.

Hyperoxia increases free radical production, leading to DNA damage. Recent studies indicate that oxygen augments the expression of p53 and p21(WAF1/CIP1), and increases apoptotic labeling of airway epithelial cells. Similar changes in regulatory gene products have not been reported in other pulmonary cells, nor have these changes been investigated in conjunction with alterations in cell-cycle distribution. The present study was conducted to determine whether oxygen alters the expression of p53 and p21(WAF1/CIP1) in human bronchial smooth-muscle cells (BSMC). BSMC placed in room air (RA), 40% O(2), or 95% O(2) were examined for 3 d to determine cell number, thymidine incorporation, cell-cycle distribution, and lactate dehydrogenase release. Apoptosis was assessed through the terminal deoxynucleotidyl transferase-deoxyuridine triphosphate end-nick labeling (TUNEL) technique, and p53 and p21(WAF1/CIP1) protein levels were determined through enzyme-linked immunosorbent assay. Exposure of BSMC to 95% O(2) decreased proliferation and DNA synthesis within 24 h, and was accompanied by an increase in S-phase cells (72 h; RA: 12.9 +/- 4.6%, versus 95% O(2): 34.6 +/- 7.0%; P < 0.01). By comparison, exposure to 40% O(2) resulted in decreased proliferation at 48 h without significant alterations in cell-cycle distribution. Both p53 and p21(WAF1/CIP1) levels were increased by 95% O(2), with maximal differences noted at 24 and 48 h, respectively. All atmospheres showed < 8% cell death and few TUNEL-positive cells. Our results indicate that oxygen-mediated alterations in BSMC proliferation are time- and concentration-dependent. Furthermore, high oxygen levels induce S-phase arrest and increased expression of p53 and p21(WAF1/CIP1). Activation of these genes may prevent replication without inducing apoptosis to allow for the repair of oxidative damage.     

Growth arrest by the LKB1 tumor suppressor: induction of p21(WAF1/CIP1)

Germline mutations of the LKB1 tumor suppressor gene lead to Peutz-Jeghers syndrome (PJS), with a predisposition to cancer. LKB1 encodes for a nuclear and cytoplasmic serine/threonine kinase, which is inactivated by mutations observed in PJS patients. Restoring LKB1 activity into cancer cell lines defective for its expression results in a G(1) cell cycle arrest. Here we have investigated molecular mechanisms leading to this arrest. Reintroduced active LKB1 was cytoplasmic and nuclear, whereas most kinase-defective PJS mutants of LKB1 localized predominantly to the nucleus. Moreover, when LKB1 was forced to remain cytoplasmic through disruption of the nuclear localization signal, it retained full growth suppression activity in a kinase-dependent manner. LKB1-mediated G(1) arrest was found to be bypassed by co-expression of the G(1) cyclins cyclin D1 and cyclin E. In addition, the protein levels of the CDK inhibitor p21(WAF1/CIP1) and p21 promoter activity were specifically upregulated in LKB1-transfected cells. Both the growth arrest and the induction of the p21 promoter were found to be p53-dependent. These results suggest that growth suppression by LKB1 is mediated through signaling of cytoplasmic LKB1 to induce p21 through a p53-dependent mechanism.