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.     

丙戊酸钠抑制A549细胞生长

目的研究丙戊酸钠对肺癌A549细胞增殖和细胞周期的影响。方法MTT检测生长抑制,流式细胞仪检测细胞周期和凋亡,Western blot检测p21WAF1/CIP1蛋白表达。结果丙戊酸钠以剂量依赖性方式抑制A549细胞生长;丙戊酸钠上调G0/G1期比例,下调S期和G2/M期,不影响细胞凋亡;丙戊酸钠上调p21WAF1/CIP1蛋白表达。结论丙戊酸钠上调p21WAF1/CIP1表达,使细胞阻滞于G0/G1期,抑制A549细胞生长。     

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。     

RUNX3 directly interacts with intracellular domain of Notch1 and suppresses Notch signaling in hepatocellular carcinoma cells

RUNX3 takes a strong suppressive effect in many tumors including hepatocellular carcinoma (HCC). HES-1, a downstream target of Notch signaling, is shown to be decreased in human HCC cell line SMMC7721 with RUNX3 gene transfection. Since Notch signaling is oncogenic in HCC, RUNX3 might exert its inhibitory effect in HCC partly through the suppression on Notch signaling. To investigate the possible mechanism of the down-regulation of HES-1 by RUNX3, we performed Western blot and reporter assay and found that RUNX3 suppressed intracellular domain of Notch1 (ICN1)-mediated transactivation of Notch signaling while it did not alter the expression of ICN1 and recombination signal binding protein-Jκ (RBP-J) in SMMC7721 cells. Besides, confocal microscopy, co-immunoprecipitation and GST pull-down assays showed that RUNX3 could co-localize with ICN1 and RBP-J, forming a complex with these two molecules in nucleus of SMMC7721 cells by its direct interaction with ICN1. Furthermore, RUNX3 was recruited to RBP-J recognition motif of HES-1 promoter, which was identified by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Taken together, these findings indicate that RUNX3 suppresses Notch signaling in HCC SMMC7721 cells by its interaction with ICN1 and thus recruitment to the RBP-J recognition motif of downstream genes of Notch signaling.     

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     

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.     

上皮调节蛋白对表皮癌细胞A431生长的抑制机制

对重组蛋白上皮调节蛋白（ｅｐｉｒｅｇｕｌｉｎ,ＥＰＩ）抑制表皮癌细胞Ａ４３１的生长机制进行了初步的探讨。通过Ｎｏｒｔｈｅｒｎ杂交发现,细胞受到重组蛋白质刺激后,其周期蛋白激酶的抑制因子ｐ２１＾ＷＡＦ１／ＣＩＰ１的ｍＲＮＡ含量有较明显的提高。报告基因分析表明,ｐ２１＾ＷＡＦ１／ＣＩＰ１启动子区的转录因子ＳＴＡＴ１结合序列对ＥＰＩ的刺激有较强的反应。流式细胞仪的测定则表明,在ＥＰＩ导致的细胞生长抑制中     

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。     

Infrequent but significant p21/WAF1/CIP1 gene alteration in synchronous or metachronous transitional cell carcinoma

p53 inducible cyclin dependent kinase inhibitor, p21/WAF1/CIP1(p21), played a pivotal role for G1 arrest when cells received genotoxic stimuli. p21 could be a putative tumor suppressor gene, since its dysfunction may lead to accumulation of genomic alteration. We investigated the p21 and p53 status using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and immunohistochemical analyses, in eight patients who had synchronous or metachronous urothelial tumors. Loss of heterozygosity (LOH) of p21 gene was detected in one coincidental tumor in one case. p21 positive cells were detected by immuno-histochemical staining in all tumors in one case, and in one coincidental tumor in two cases. Among p21 positive cells in these three cases, no p53 mutations were detected, whereas no p21 positive cells were detected in other cases with a p53 mutation. These findings suggested that in transitional cell carcinoma (TCC) p21 gene mutation is infrequent like the p53 gene mutation, but that LOH might be important in the inactivation of p21.