抑癌基因RB-蛋白与肿瘤相关的研究进展

RB基因是人类最早发现和确定的抑癌基因,表达产物pRb是重要的细胞周期调节因子,参与细胞的生长分化。pRb功能的缺陷或丧失是很多肿瘤发生的基础,正常RB基因导入恶性肿瘤细胞能全部或部分抑制其恶性表现,也能增加常规化疗和放疗的效果,经截短修饰的RB94在体内和体外都有更好的抑癌作用。但pRb同时也被证实具有保护细胞免于凋亡的作用,此点与其抑癌作用相悖,其各方面功能也可受MicroRNA分子的调控。正是由于RB功能的多面性,近年来对RB与肿瘤的发生、发展和抑制作用的研究从未间断,不断有新的疑问和功能路径被发现。     

Low- and high-risk human papillomavirus E7 proteins regulate p130 differently

The E7 protein of high-risk human papillomaviruses (HR HPVs) targets pRb family members (pRb, p107 and p130) for degradation; low-risk (LR) HPV E7 only targets p130 for degradation. The effect of HR HPV 16 E7 and LR HPV 6 E7 on p130 intracellular localization and half-life was examined. Nuclear/cytoplasmic fractionation and immunofluorescence showed that, in contrast to control and HPV 6 E7-expressing cells, a greater amount of p130 was present in the cytoplasm in the presence of HPV 16 E7. The half-life of p130, relative to control cells, was decreased in the cytoplasm in the presence of HPV 6 E7 or HPV 16 E7, but only decreased by HPV 6 E7 in the nucleus. Inhibition of proteasomal degradation extended the half-life of p130, regardless of intracellular localization. These results suggest that there may be divergent mechanisms by which LR and HR HPV E7 target p130 for degradation.     

Analysis of the Cycilin D1/p16/pRb Pathway in Parathyroid Adenomas

Cyclin D1/p16/pRb pathway controlling G1→S cell cycle checkpoint is frequently altered in human tumors. Currently, scarce data are available for parathyroid tumors. We have studied 46 parathyroid adenomas (PTAs) and 12 normal parathyroid glands (PTGs) by immunohistochemistry with cyclin D1 (CD1), p16, pRb, and Ki-67 antibodies. We observed CD1 expression in 89%, p16 in 70%, and pRb in 100% of PTAs. Statistically significant differences with normal PTGs were found only concerning p16 expression (p<0.05). Proliferating rate (Ki-67) was always low, although significantly higher than in normal PTGs. Our findings demonstrate the presence of alterations in the CD1/p16/pRb pathway in PTAs, consisting in p16 overexpression apparently unrelated to pRb downregulation. On the other hand, we did not find significant differences in CD1 expression between PTAs and normal PTGs, suggesting CD1 overexpression could be a physiological event in parathyroid tissue.     

The prognostic value of p16 and p53 expression for survival after vulvar cancer: A systematic review and meta-analysis

The tumor suppressor proteins p16 and p53 have been suggested to have prognostic value in some human papillomavirus (HPV)-associated cancers, however, this has been less well established for vulvar cancer. The aim of this review and meta-analysis was to examine the prognostic value of p16 and p53 expression status on survival after vulvar squamous cell carcinoma (VSCC). We conducted a thorough systematic literature search of multiple databases to identify studies examining survival after histolocally verified VSCC that were tested for p16 and/or p53. A total of 18 eligible studies were included. Using a fixed-effects model we calculated study-specific and pooled hazard ratios (HRs) of 5-year overall survival (OS). In the analyses of OS, we included 475 VSCC cases tested for p16 expression of which 38% were p16 positive. The pooled HR_p16 was 0.40 (95% CI: 0.29–0.55). In addition, the majority of results from studies with adjusted analyses on the prognostic value of p16 indicated that p16 expression status could be an independent prognostic marker for OS in women diagnosed with VSCC, and the same pattern was seen for disease specific survival (DSS). We also included 310 VSCC cases tested for p53 expression of which 54% were p53 positive. The pooled HR_p53 was 1.81 (95% CI: 1.22–2.68) indicating that p53 positive VSCC have a significantly lower 5-year OS compared to p53 negative. The results in relation to p53 reported from adjusted analyses OS and on DSS and disease free survival were more equivocal. This meta-analysis and review suggests that p53 and especially p16 expression status are of prognostic importance in women diagnosed with VSCC. This may be clinically important in the future design of targeted therapy and when planning the optimal follow-up strategy. Future studies should include the combined use of biomarkers such as p16, p53 and HPV status.     

Enforced expression of the tumor suppressor p53 renders human leukemia cells (U937) more sensitive to 1-[beta-D-arabinofuranosyl]cytosine (ara-C)-induced apoptosis

The effects of enforced expression of p53 on the sensitivity of p53(-/-) human monocytic leukemia cells (U937) to apoptosis following exposure to the S-phase-specific antimetabolite 1-[beta-D-arabinofuranosyl]cytosine (ara-C) were examined. Cells were stably transfected with a plasmid containing a chimeric DNA construct encoding a temperature-sensitive p53 variant (135(ala-->val)), which transactivates at 32 degrees but is non-functional at 37 degrees. A significant reduction in the S-phase population was observed in ptsp53 mutants incubated at 32 degrees. Nevertheless, while vector controls did not exhibit differential sensitivity to ara-C at 32 degrees versus 37 degrees, temperature-sensitive p53 mutants displayed a significant increase in apoptosis at the permissive temperature. This was not accompanied by increased ara-CTP formation, DNA incorporation of [3H]ara-C, or altered expression of Bcl-2 or Bax. Enhanced sensitivity was associated with increased mitochondrial injury (e.g. cytochrome c release), caspase activation, and loss of clonogenic survival. Significantly, ptsp53 cells synchronized in S phase were markedly more sensitive to ara-C-mediated mitochondrial injury and apoptosis at 32 degrees, indicating that wild-type p53 specifically enhances the susceptibility of this subpopulation to ara-C lethality. Consistent with these results, transient transfection of human wild-type p53 cDNA rendered parental U937 cells more sensitive to ara-C-mediated cell death. Collectively, these findings indicate that p53 expression renders S-phase U937 cells more susceptible to ara-C-mediated mitochondrial dysfunction, cytochrome c release, apoptosis, and loss of clonogenic survival without enhancing ara-C metabolism. Such findings raise the possibility that loss of functional p53 activity allows leukemia cells to circumvent ara-C lethality.     

PCNA和pRb2/p130在子宫内膜癌中的表达及临床意义

目的：探讨增殖细胞抗原（ PCNA）和pRb2/p130在正常子宫内膜、子宫内膜增殖症、非典型子宫内膜增殖症和子宫内膜癌中的表达、相关性及与临床病理特征之间的关系。方法：免疫组织化学EnVision二步法检测PCNA和pRb2/p130在30例正常增生期子宫内膜、30例子宫内膜增殖症、40例非典型增生子宫内膜增殖症和76例子宫内膜癌中的表达。结果：PCNA在非典型子宫内膜增殖症、子宫内膜癌中的表达分别高于正常子宫内膜和子宫内膜增殖症,差异显著（ P＝0.043;P＝0.020）,pRb2/p130的表达分别低于正常子宫内膜和子宫内膜增殖症,差异显著（ P﹤0.05）;子宫内膜癌中,PCNA与肿瘤大小、分期、淋巴结转移和雌激素差异显著（P﹤0.05）,PCNA在子宫内膜癌中的表达显著高于子宫内膜增殖症（P＝0.045）,pRb2/p130与年龄、组织学分级、浸润深度、淋巴结转移和雌激素有差异（ P﹤0.05）,pRb2/p130和PCNA呈负相关（ r＝-0.331,P＝0.003）。结论：pRb2/p130和PCNA在非典型子宫内膜增殖症和子宫内膜癌中的异常表达,结合在一起可能成为评估识别具有高风险的子宫内膜癌患者的新参数。     

口腔鳞癌中p16/CDK4/cyclinD1/pRb通路的表达及其意义

目的分析口腔黏膜鳞状细胞癌(OSCC)中pRb、CDK4c、yclinD1、p16INK4a等细胞周期调控因子的表达状况和相互间的联系及其临床意义。方法采用免疫组化SP法,研究47例OSCC及10例正常口腔黏膜中pRb、CDK4c、yclinD1和p16INK4a的表达情况,并结合随访资料进行相关性分析。结果47例OSCC中,pRb、CDK4、cyclinD1和p16INK4a阳性表达率分别为55%、60%、74%和38%,与正常口腔黏膜中的表达有显著差异(P<0.05)。cyclinD1的表达和淋巴结转移有密切关系(P<0.05),p16和pRb呈负相关(r=-0.312)。结论p16/Rb通路蛋白的异常表达和OSCC的发生有密切的关系;cyclinD1可作为OSCC预后的辅助性指标之一。     

Alterations of the Cell Cycle Regulators Cyclin D1, Cyclin A, p27, p21, p16, and pRb in Apocrine Metaplasia of the Breast

Abstract:  G1/S transition defects have been a proposed requirement for tumor development. Apocrine metaplasia (APM) in the breast has been held as a sign of benignity. Yet, a number of studies have reported the presence of molecular abnormalities in some forms of APM suggesting a possible oncogenic potential for some of these lesions. We currently investigate the role of some of the cell cycle proteins, previously reported to be de-regulated in breast cancer, in an attempt to assess their significance in APM. Using immunohistochemistry, the expression of cyclin D1, cyclin A, p27, p21, p16, pRb and Ki-67 was examined in 93 cases of APM. The cases were divided into nonpapillary (NAPM) (30 cases) and papillary metaplasia (PAPM) (63 cases). PAPM was further subdivided into simple papillary (SPAPM) (29 cases), complex papillary (28 cases) and highly complex papillary (six cases). For statistical analysis, the last two groups were merged together (CPAPM). The results showed that increased histological complexity was associated with significant increase of proliferative capacity and alterations of the cell cycle control. The median Ki-67 index was 1.5% in SPAPM and 4.8% in the CPAPM. Also, alterations of the cell cycle regulators were significantly higher in the CPAPM than in the SPAPM. NAPM was generally similar to normal breast epithelium. Apocrine cells were negative for p16 while pRb was expressed in all cases. These findings suggest that in complex forms of APM, there is a considerable degree of cellular unrest. This may contribute to increased susceptibility to carcinogenesis.     

鱼藤素对淋巴瘤Daudi细胞株细胞增殖细胞凋亡的影响及其机制

目的观察鱼藤素对人Burkitt淋巴瘤Daudi细胞株细胞增殖、细胞凋亡和细胞周期的影响,并探讨其分子机制。方法四甲基偶氮唑蓝(MTY)法检测细胞增殖活性,Hoechst 33258染色和Annexin-V／PI双标法检测细胞凋亡,流式细胞仪检测细胞周期分布,Western blot检测细胞内cyclin D1和pRb的蛋白表达。结果鱼藤素对Daudi细胞具有明显的增殖抑制作用,而对正常人外周血单个核细胞(PBMC)抑制作用不明显。鱼藤素可以诱导Daudi细胞凋亡,Hoechst 33258染色可见典型凋亡小体。Annexin V／PI双标法显示,鱼藤素诱导细胞发生早期凋亡,并呈剂量依赖性,20、40、80 nmol／L鱼藤素作用24 h时,凋亡率分别为15．46％±0．62％、18．48％±2．98％和31．42％±1．43％。鱼藤素作用Daudi细胞后,主要使细胞周期聚积于G0／G1期,G0／G1期细胞比例随鱼藤素剂量增大而逐渐增高,40 nmol／L鱼藤素作用24 h达56．56％;相反,S期细胞比例随鱼藤素剂量增大而逐渐降低,对G2／M期细胞作用不明显。鱼藤素使cyclin D1及pRb蛋白表达降低,呈剂量依赖关系。结论鱼藤素抑制Daudi细胞增殖,使细胞阻滞于G0／G1期,并诱导细胞凋亡。其抗肿瘤机制可能与下调cyclin D1和pRb蛋白表达有关。     

蟾蜍灵对胃癌组织MGC-803细胞周期作用机制的研究

目的探讨蟾蜍灵对胃癌MGC803细胞周期及周期蛋白的影响。方法采用台盼蓝拒染法测定细胞增殖活力;瑞士姬姆萨染色观察细胞形态学的变化;流式细胞仪检测细胞周期;免疫组织化学方法检测细胞周期相关蛋白p16、p21、pRb的表达。结果1)蟾蜍灵抑制胃癌MGC803细胞的增殖,24、48、72h的IC50分别为0.086、0.048和0.036μmol/L。2)蟾蜍灵在0.01～0.1μmol/L时作用24～72h,形态学上可见细胞体积增大,出现双核、多核细胞。3)流式细胞仪显示蟾蜍灵浓度为0.01～0.1μmol/L时可明显诱导细胞周期G2/M期阻滞。4)蟾蜍灵作用于胃癌MGC803细胞,观察到p16、p21、pRb蛋白的表达明显上调。结论蟾蜍灵引起胃癌MGC803细胞的周期阻滞可能与p16、p21、pRb蛋白的上调有关。