p21WAFl/CIPl、PCNA在骨巨细胞瘤中的表达及意义

目的探讨p21WAF1/CIP1、PCNA在骨巨细胞瘤中的表达特点及其与骨巨细胞瘤的分化和复发的关系.方法应用LSAB免疫组织化学方法对38例骨巨细胞瘤中的p21WAF1/CIP1、PCNA的表达进行检测.结果66.8%的骨巨细胞瘤中可检测到p21WAF1/CIP1的阳性表达,其阳性表达主要位于分化好的多核巨细胞的细胞核内,分化好的骨巨细胞瘤(Ⅰ级)中p21WAF1/CIP1的表达明显高于分化差组(Ⅱ、Ⅲ级)(P<0.01).38例骨巨细胞瘤均可检测到PCNA的阳性表达,其阳性表达主要位于单核基质细胞的细胞核内;未复发组及复发组中p21WAF1/CIP1、PCNA的表达无显著性差异(P>0.05).结论骨巨细胞瘤中p21WAF1/CIP1的表达与骨巨细胞瘤的分化相关,可作为骨巨细胞瘤分化的参考指标.     

Mitochondrial ribosomal protein S36 delays cell cycle progression in association with p53 modification and p21(WAF1/CIP1) expression

Ribosomal biogenesis is correlated with cell cycle, cell proliferation, cell growth and tumorigenesis. Some oncogenes and tumor suppressors are involved in regulating the formation of mature ribosome and affecting the ribosomal biogenesis. In previous studies, the mitochondrial ribosomal protein L41 was reported to be involved in cell proliferation regulating through p21(WAF1/CIP1) and p53 pathway. In this report, we have identified a mitochondrial ribosomal protein S36 (mMRPS36), which is localized in the mitochondria, and demonstrated that overexpression of mMRPS36 in cells retards the cell proliferation and delays cell cycle progression. In addition, the mMRPS36 overexpression induces p21(WAF1/CIP1) expression, and regulates the expression and phosphorylation of p53. Our result also indicate that overexpression of mMRPS36 affects the mitochondrial function. These results suggest that mMRPS36 plays an important role in mitochondrial ribosomal biogenesis, which may cause nucleolar stress, thereby leading to cell cycle delay.     

DNA损伤生物学反应中ATM对p21~(WAF1/CIP1)蛋白的直接磷酸化

毛细血管扩张性共济失调症突变蛋白 (mutatedinataxiatelangiectasia ,ATM)是直接感受DNA双链断裂损伤并起始诸多DNA损伤信号反应通路的主开关分子 .已有研究发现 ,DNA损伤生物学反应中 ,ATM可通过磷酸化活化p5 3,继而转录活化细胞周期检查点蛋白p2 1WAF1 CIP1的表达 ,而对于ATM是否直接参与p2 1WAF1 CIP1的早期活化迄今尚无实验证明 .通过免疫共沉淀反应 ,检测到细胞电离辐射 (ionizingradiation ,IR)反应早期ATM与p2 1WAF1 CIP1蛋白存在相互作用 .将p2 1WAF1 CIP1蛋白编码基因全长克隆入原核表达载体pGEX4T 2 ,经诱导表达及亲和层析纯化获取GST p2 1融合蛋白作为磷酸化底物 .体外磷酸化实验检测证明 ,IR活化的ATM具磷酸化p2 1WAF1 CIP1蛋白的功能 ,并且此磷酸化功能可被PI3K家族特异性抑制剂Wortmannin所抑制 .结果揭示了IR后ATM可通过直接磷酸化p2 1WAF1 CIP1蛋白 ,在IR致DNA损伤生物学反应早期调控p2 1WAF1 CIP1蛋白的快速活化过程     

利用RNA干扰抑制p21WAF1/CIP1基因的表达及周期阻滞效应

目的：构建p21WAF1/CIP1基因小干扰RNA（siRNA）的真核表达载体,观察其对p21WAF1/CIP1表达的影响和细胞周期的变化。方法：合成了针对p21WAF1/CIP1基因的siRNA,将其克隆到siRNA表达载体pSliencer2.1-U6neo上,将重组质粒和带FLAG标签的p21WAF1/CIP1共转染293T人胚肾细胞,通过Westernblot检验RNA干扰（RNAi）敲低外源p21WAF1/CIP1的效果;将重组质粒单独转染293T人胚肾细胞,利用p21WAF1/CIP1抗体检测RNAi敲低内源p21WAF1/CIP1的效果;利用流式细胞仪检测敲低后细胞周期的变化。结果：测序证明构建了p21WAF1/CIP1siRNA真核表达载体;Westernblot和流式细胞分析证明,构建的siRNA能有效降低p21WAF1/CIP1基因的表达,并且使G1期细胞数减少14.03%,S期细胞增多13.45%。结论：构建了p21WAF1/CIP1siRNA的真核表达载体,该siRNA能有效抑制p21WAF1/CIP1基因的表达并部分解除了G1期阻滞。     

姜黄素对肝癌细胞HepG2的抗癌作用及P21WAF1/CIP1表达的影响

目的：探讨姜黄素对肝癌HepG2细胞抗癌作用及相关周期蛋白依赖激酶抑制因子P21WAF1/CIP1表达的影响。方法：体外培养肝癌HepG2细胞,用MTT法检测姜黄素对HepG2细胞的抑制作用,以RT-PCR方法检测HepG2细胞中P21WAF1/CIP1mRNA的表达,用免疫细胞化学检测其P21WAF1/CIP1蛋白的表达。结果：姜黄素呈时间剂量性抑制HepG2细胞的生长,并显著上调HepG2细胞中P21WAF1/CIP1mRNA和蛋白的表达。结论：姜黄素能抑制HepG2细胞的生长,并上调其中P21WAF1/CIP1的表达。     

姜黄素对肝癌细胞HepG2 的抗癌作用及P21WAF1/CIP1 表达的影响

目的:探讨姜黄素对肝癌HepG2细胞抗癌作用及相关周期蛋白依赖激酶抑制因子P21WAF1/CIP1表达的影响.方法:体外培养肝癌HepG2细胞,用MTT法检测姜黄素对HepG2细胞的抑制作用,以RT-PCR方法检测HepG2细胞中P21WAF1/CIP1mRNA的表达,用免疫细胞化学检测其P21WAF1/CIP1蛋白的表达.结果:姜黄素呈时间剂量性抑制HepG2细胞的生长,并显著上调HepG2细胞中P21WAF1/CIP1mRNA和蛋白的表达.结论:姜黄素能抑制HepG2细胞的生长,并上调其中P21WAF1/CIP1的表达.     

Adriamycin (ADM) induced apoptosis in Transitional Cell Cancer (TCC) cell lines accompanied by p21 WAF1/CIP1 induction

Genotoxic stimuli, including anticancer drugs, induce apoptosis in cancer cells through increase of p53, p21WAF1/CIP1 , at least in part. Bcl-2 and Bax modify this pathway or directly regulated by p53. Here we studied Adriamycin (ADM)-induced apoptosis in four human bladder cancer cell lines in respect of p53, p21WAF1/CIP1 and Bcl-2 family proteins. After ADM, treatment bladder cancer cells underwent dose-dependent cell death with typical morphologic features of apoptosis. Among four cell lines RT4 with wt p53, low ratio of Bcl-2 to Bax and induction of p21WAF1/CIP1 after ADM treatment, was the most sensitive to induction of apoptosis. Thus, p53, p21WAF1/CIP1 , Bcl-2 and Bax status might determine susceptibility of bladder cancer cells to ADM induced apoptosis.     

HeLa细胞中Skp2的表达调控p21WAFCIP1稳定性

泛素 蛋白酶体抑制剂MG 132处理的HeLa和SaoS 2细胞中 ,低表达的p2 1WAF CIP1受泛素 蛋白酶体通路调控 .为探讨肿瘤细胞中高表达的E3连接酶底物结合亚基 Skp2和低表达的p2 1WAF CIP1之间的关系 ,在HeLa细胞中转染Skp2的反义寡核苷酸后 ,p2 1表达水平明显升高 .同时 ,相对于转染空载体和Skp2ΔF(Skp2的F box缺失突变体 )的真核表达载体 ,转染全长Skp2的HeLa细胞中 ,p2 1表达水平显著下降 ,说明肿瘤细胞中Skp2调节p2 1的稳定性 ,并且这种调控作用依赖于Skp2蛋白中的F box结构 .免疫荧光结果表明 ,Skp2和p2 1共定位于细胞核 ,这为两者间的相互作用提供了前提 ;体内相互免疫共沉淀结果表明 ,p2 1和其它SCFSkp2 的底物一样与Skp2直接结合 ,并且它们之间的结合区不在F box结构域 ,这一结果在体外的GST pulldown实验中得到验证     

PKCdelta modulates p21WAF1/CIP1 ability to bind to Cdk2 during TNFalpha-induced apoptosis

Cyclin-dependent kinase 2 (Cdk2) activity is thought to be involved in cell death-associated chromatin condensation and other manifestations of apoptotic death. Here we show that during TNFalpha-induced apoptosis, PKCdelta is activated in a caspase-3-dependent manner and phosphorylates p21(WAF1/CIP1), a specific cyclin-dependent kinase inhibitor, on (146)Ser. This residue is located near a cyclin-binding motif (Cy2) that plays an important role in the interaction between p21(WAF1/CIP1) and Cdk2, and its phosphorylation modulates the ability of p21(WAF1/CIP1) to associate with Cdk2. The phosphorylation of p21(WAF1/CIP1) is temporally related to the activation kinetics of Cdk2 activity during the apoptosis. We propose that during TNFalpha-induced apoptosis, PKCdelta-mediated phosphorylation of p21(WAF1/CIP1) at (146)Ser attenuates the Cdk2 binding of p21(WAF1/CIP1) and thereby upregulates Cdk2 activity.     

Mitochondrial ribosomal protein L41 mediates serum starvation-induced cell-cycle arrest through an increase of p21(WAF1/CIP1)

Ribosomal proteins not only act as components of the translation apparatus but also regulate cell proliferation and apoptosis. A previous study reported that MRPL41 plays an important role in p53-dependent apoptosis. It also showed that MRPL41 arrests the cell cycle by stabilizing p27(Kip1) in the absence of p53. This study found that MRPL41 mediates the p21(WAF1/CIP1)-mediated G1 arrest in response to serum starvation. The cells were released from serum starvation-induced G1 arrest via the siRNA-mediated blocking of MRPL41 expression. Overall, these results suggest that MRPL41 arrests the cell cycle by increasing the p21(WAF1/CIP1) and p27(Kip1) levels under the growth inhibitory conditions.     

胃癌组织中p53、p21WAF1蛋白的表达及与幽门螺杆菌L型感染的关系研究

目的:研究胃癌组织中磷蛋白53(P53)、磷蛋白21野生型p53活化片段(p21WAF1)蛋白的表达及与幽门螺杆菌L型(Hp-L)感染的关系。方法:选择我院2013年1月至2017年1月病理确诊为胃癌的标本220例为胃癌组,另外选取胃癌组织旁的健康组织为对照组。Hp-L采用快速尿素酶测试法、Giemsa染色法检测,P53、p21WAF1阳性表达采用HE法及免疫组化法测定,并采用Spearman等级检验分析其相关性。结果:胃癌组p53阳性表达率显著高于对照组(P0.05);p21WAF1阳性表达率显著低于对照组(P0.05),胃癌组织中p53和p21WAF1表达呈负相关(P0.05)。胃癌组Hp-L阳性感染率显著高于对照组(P0.05);胃癌组Hp-L阳性组p53阳性表达率显著高于于阴性组(P0.05);p21WAF1阳性表达率显著低于阴性组(P0.05),胃癌组织中p53与Hp-L阳性表达呈正相关(P0.05),p21WAF1与Hp-L阳性表达呈负相关(P0.05),p53阳性表达仅与浸润程度有关(P0.05),P21WAF1表达与病理分级、浸润程度、UICC分期有关(P0.05)。结论:Hp-L是胃癌发生的主要诱因,癌症发展过程中,Hp-L型感染可上调p53表达、抑制p21WAF1表达,且p53、p21WAF1的表达变化与细胞浸润程度有密切联系,对胃癌的临床治疗具有重要作用。     

8-chloroadenosine induced HL-60 cell growth inhibition, differentiation, and G(0)/G(1) arrest involves attenuated cyclin D1 and telomerase and up-regulated p21(WAF1/CIP1)

8-Chloroadenosine, an active dephosphorylated metabolite of the antineoplastic agent 8-chloroadenosine 3',5'-monophosphate (8-Cl-cAMP), induces growth inhibition in multiple carcinomas. Here we report that 8-chloroadenosine inhibits growth in human promyelocytic leukemia HL-60 cells by a G(0)/G(1) phase arrest and terminates cell differentiation along the granulocytic lineage. The mechanism of 8-chloroadenosine-induced G(0)/G(1) arrest is independent of apoptosis. The expressions of cyclin D1 and c-myc in HL-60 are suppressed by 8-chloroadenosine, whereas the cyclin-dependent kinases inhibitor p21(WAF1/CIP1) is up-regulated. 8-Chloroadenosine has less effect on the expressions of cyclin-dependent kinase (cdk)2 and cdk4, G(1) phase cyclin-dependent kinases, and only moderately induces the expression of transforming growth factor beta1 (TGFbeta1) and the mitotic inhibitor p27(KIP1). Telomerase activity is reduced in extracts of 8-chloroadenosine treated HL-60 cells, but 8-chloroadenosine does not directly inhibit the catalytic activity of telomerase in vitro. Therefore, anti-proliferation of HL-60 cells by 8-chloroadenosine involves coordination of cyclin D1 suppression, reduction of telomerase activity, and up-regulation of p21(WAF1/CIP1) that arrest cell-cycle progression at G(0)/G(1) phase and terminate cell differentiation.     

外源p21~(WAF1)转染对人成纤维细胞凋亡的影响

构建了有义和反义ｐ２１ＷＡＦ１ 逆转录病毒表达载体, 分别经脂质体包裹后转染人胚肺二倍体成纤维细胞（２ＢＳ）。Ｓｏｕｔｈｅｒｎ 印迹杂交证实转染细胞中外源ｐ２１ ＷＡＦ１ｃＤＮＡ 已整合入基因组中。与空载体转染细胞相比, 有义转染细胞的ｐ２１ＷＡＦ１ ｍ ＲＮＡ 表达上升; 细胞增殖速度明显减慢; 对丁酸钠诱导凋亡的敏感性降低, 表现在细胞存活率升高, 核ＤＮＡ 梯状断裂片段出现的时间滞后, 断裂片段浓度下降, 流式细胞计检测的凋亡峰面积缩小。而反义转染细胞的ｐ２１ＷＡＦ１ ｍ ＲＮＡ表达下降; 细胞增殖速度较快; 对丁酸钠诱导凋亡的敏感性上升, 有关表现与有义转染细胞相反。说明２ＢＳ细胞内ｐ２１ＷＡＦ１ 的表达量与其被丁酸钠诱导凋亡的能力呈负相关。     

p21WAF1在丁酸钠诱导的人成纤维细胞凋亡中的表现

用丁酸钠 (NaBu)诱导了人胚肺二倍体成纤维细胞凋亡 (2BS) ,检测其诱导过程中凋亡相关基因的表达变化 ,结果表明 ,p2 1WAF1的表达在凋亡发生前即有明显下降 ,并持续至凋亡发生时 ,bcl 2的表达仅在凋亡发生时有所下降 ,c myc和c fos的表达有所上升 ,而 p5 3和HER 2的表达无明显变化 .用稳定转染了不同长度p2 1WAF1启动子片段和下游绿色荧光蛋白 (GFP)报告基因的 2BS WP系列细胞进一步研究发现 ,其GFP的表达水平在NaBu诱导过程中下降 ,主要调控区域为 p2 1WAF1启动子的TATAbox上游 0～ - 80 0bp .说明NaBu诱导的人胚肺二倍体成纤维细胞凋亡与p2 1WAF1启动子的转录活性下降与密切相关 ,并且可能不依赖于p5 3.