ARF——一种新的抑癌因子的研究进展

INK4a/ARF基因位于人染色体9p21,是人类肿瘤中最常见的基因失活位点之一.INK4a/ARF基因有两套各自独立的启动子,通过可变阅读框,能够编码两种蛋白质：p16^INK4a和p14^ARF（ARF在鼠细胞中为p19^ARF）.p16作为CDK4/6的抑制因子,能够阻断pRb磷酸化,将细胞周期阻断在G1期;而ARF可结合原癌蛋白MDM2,稳定p53,将细胞周期阻断在G1期和G2/M转换期,或诱导细胞凋亡.因此ARF蛋白和p16一样也是一种肿瘤抑制因子.     

p14ARF对人黑色素瘤细胞增殖的影响及其作用机理的初探

ARF(alternative reading frame)作为INK4a/ARF的β转录产物,能够稳定p53, 诱导细胞周期阻断或凋亡.利用高表达p14^ARF的人黑色素瘤细胞模型,探讨了ARF抑制细胞增殖的分子作用机理.研究发现p14^ARF高表达能将细胞周期阻断在G1和G2期, p53, p21^cip1和p27^kip1蛋白水平明显增强, 而p-ERK1/2,CyclinD1和CyclinE蛋白水平下降, 明显抑制细胞生长. 提示p14^ARF能通过ERK(extracellular signal-regulated kinase)信号通路相互协调作用抑制A375细胞增殖.     

细胞周期抑制因子p19ARF对人二倍体细胞衰老的影响

为了探讨细胞周期抑制因子p19ARF对人二倍体细胞复制性衰老的影响, 构建了重组p19ARF真核表达载体, 并通过脂质体的介导将p19ARF基因转染到人二倍体成纤维细胞WI-38中过表达, 观察其对WI-38细胞衰老的影响. 结果发现与对照细胞相比, 在p19ARF基因导入后, 细胞中p53和p21的表达水平明显上调, 细胞传代数减少10~12代, 生长速率降低, 细胞周期阻滞于G₁期, 衰老标志物SA-β-gal染色阳性率上升, 线粒体膜电位下降, 细胞形态呈衰老细胞样变化, 这些结果表明p19ARF高表达可促进人二倍体细胞的衰老进程.     

p16INK4a基因的功能及其调控

p16^INK4a蛋白能抑制CDK4和CDK6的活性,使pRb处于非磷酸化或低磷酸化状态而能与转录因子E2Fs结合,从而抑制DNA 的合成,阻止细胞由G1期进入S期.p16^INK4a的表达受Ets1和Ets2的正调控,受Bmi-1的负调控.p16^INK4a基因缺失、突变、甲基化、RNA剪接加工错误可导致细胞周期失控和癌变.应用p16^INK4a对某些肿瘤进行基因治疗的研究正在进行中.     

可逆的组蛋白乙酰化调控p16的表达

p16^INK4a通过抑制CDK4/6的活性而在细胞周期进行中发挥重要的作用,研究发现,组蛋白乙酰转移酶p300能促进p16^INK4a启动子活性,而组蛋白去乙酰化酶HDAC3/4能够逆转由p300介导的p16^INK4a启动子活性的增加,HDAC3/4能够降低p16^INK4a mRNA和蛋白质的水平．染色质免疫沉淀(ChIP)实验结果表明转染p300表达质粒能够逆转由HDAC3/4介导的p16^INK4a启动子组蛋白的低乙酰化状态．此外,免疫荧光实验结果表明HDAC4的核质穿梭起着重要的作用．免疫印迹和染色质免疫沉淀实验证明HDAC的抑制剂丁酸钠盐(NaBu)能通过诱导组蛋白的高乙酰化而促进p16^INK4a的表达．基于这些实验结果,推测出可逆的组蛋白乙酰化参与p16^INK4a基因转录调控的模型．     

细胞周期相关蛋白cyclin D1、p53和p21WAF1/Cip1在食管鳞癌中的表达改变及其病理学意义

食管鳞状细胞癌(Esophageal squamous cell carcinoma, ESCC)是我国常见的恶性肿瘤之一, 虽然临床诊治手段正逐步改进, 但中晚期患者5年生存率仍然很低。目前认为细胞周期调控异常与肿瘤发生发展关系密切, 然而相关周期调节蛋白在食管癌患者中的表达改变、临床意义及其应用价值还没有明确结论。文章应用组织微阵列联合免疫组织化学技术(TMA-IHC), 对148例食管鳞癌组织标本中细胞G₁/S期调控蛋白cyclin D1、p53和p21^WAF1/Cip1的表达进行检测, 分析其与临床病理参数之间的相关性。结果显示, cyclin D1与p53蛋白在食管癌细胞中表达升高, p53表达阳性率与区域淋巴结转移显著相关(P = 0.001)。p21^WAF1/Cip1蛋白在肿瘤组织中表达降低, 且p21WAF1/Cip1表达阴性患者的术后生存时间显著短于表达阳性的患者(P = 0.001)。多因素生存分析显示p21WAF1/Cip1是一个独立的预后因素(相对危险度为0.418, P<0.001)。微阵列比较基因组杂交(array-CGH)检测进一步表明45.4%的食管癌患者存在cyclin D1基因扩增。以上结果提示食管鳞癌中存在细胞周期G1/S期调控异常, p21^WAF1/Cip1蛋白可能是一个有应用价值的预后因子。     

贮存式自体成分输血对胃肠肿瘤根治术患者T淋巴细胞亚群、血液流变学和预后的影响

摘要 目的：研究贮存式自体成分输血对胃肠肿瘤根治术患者T淋巴细胞亚群、血液流变学以及预后的影响。方法：选取2016年12月~2017年12月我院收治的95例行胃肠肿瘤根治术的胃肠肿瘤早期患者作为研究对象。按随机数表法分为A组（n=47,贮存式自体成分输血）和B组(n=48,异体成分输血)。比较两组患者血常规指标[红细胞计数(RBC)、血红蛋白（Hb）、血细胞比容（Hct）]、免疫功能指标（CD3⁺、CD4⁺、CD8⁺、CD4⁺/CD8⁺）、血液流变学指标[红细胞沉降率、平均血液黏度、红细胞刚性指数及红细胞变形指数]的变化。随访患者2年,采用Kaplan-Meier曲线分析两组预后情况。结果：术前至术后7 d 时间段A组的CD3⁺、CD4⁺水平及CD4⁺/CD8⁺呈先降低后升高趋势,且术后7 d已恢复至术前水平;B组术后1 d、3 d、7 d的CD3⁺、CD4⁺水平及CD4⁺/CD8⁺明显低于A组(P＜0.05),两组CD8⁺水平相比无差异(P＞0.05) 。两组术后7 d的RBC、Hct、Hb分别较输血前下降（P＜0.05）,但两组间比较差异无统计学意义(P＞0.05);两组患者术后7 d的红细胞沉降率均升高,但A组低于B组(P＜0.05) ,两组患者术后的红细胞变形指数、平均血液黏度和红细胞刚性指数比较无差异 (P＞0.05) 。Kaplan-Meier检验结果显示,A组较B组生存率明显升高（P＜0.05）。结论：贮存式自体成分输血对胃肠肿瘤根治术患者红细胞沉降率、细胞免疫功能的影响程度较异体成分输血减轻,且贮存式自体成分输血可以明显提高患者的术后生存率。     

鸟氨酸脱羧酶活性微量测定法

本文采用一种简单,微量反应系统,根据 ¹⁴C-鸟氨酸释放的 ¹⁴CO₂量测定鸟氨酸脱羧酶(ODC)的活性,酶反应在置于液闪计数瓶内的玻璃小管中进行,释放的 ¹⁴CO₂被瓶内滤纸片上的海胺吸收。实验结果表明,加酸释放 ¹⁴CO₂后30分钟 ¹⁴CO₂吸收已达最大值,且吸收量与释放量成正比,酶反应测定证明 ¹⁴CO₂释放速度在40分钟内保持恒定。ODC活性与酶浓度呈线性关系,此方法不仅用于ODC活性测定,而且亦可用于其他脱羧酶活性的测定。     

黄芪多糖基于PI3K/Akt通路对食管癌大鼠抑瘤作用及免疫功能的影响

摘要 目的：探究黄芪多糖（APS）对食管癌（EPC）模型大鼠的抑瘤作用、免疫功能及磷脂酰肌醇-3-激酶（PI3K）/蛋白激酶B（Akt）信号通路的影响。方法：选取6周龄SPF级健康SD大鼠50只,随机分为模型组(M组)、顺铂组（S组）、黄芪多糖低剂量组（APSL）、黄芪多糖中剂量组（APSM）、黄芪多糖高剂量组（APSH）,每组10只。通过移植人食管癌Eca109细胞建立EPC模型,分别给予3 mg/kg顺铂和100、200、400 mg/kg的APS干预。测量各组大鼠肿瘤体积和肿瘤质量,计算肿瘤生长抑制率、胸腺指数和脾指数,HE染色观察肿瘤组织形态学,采用流式细胞仪检测外周血CD3⁺、CD4⁺、CD8⁺T淋巴细胞群,Western blot测定各组肿瘤组织中PI3K和Akt蛋白的相对表达。结果：经干预后,S组和各APS组大鼠肿瘤体积和肿瘤质量均显著小于M组（P＜0.05）;S组和APSH组肿瘤体积和肿瘤质量相显著小于APSM和APSL组（P＜0.05）;APSM组大鼠肿瘤体积和肿瘤质量显著小于APSL组（P＜0.05）;APSH、APSM和APSL组的抑瘤率分别为45.59%、32.35%和17.65%。S组大鼠的胸腺指数和脾指数均显著低于M组（P＜0.05）;各APS组大鼠的胸腺指数和脾指数均显著高于S组（P＜0.05）;APSH和APSM组大鼠胸腺指数和脾指数均显著高于M组（P＜0.05）。各APS组大鼠CD3⁺、CD4⁺和CD4⁺/CD8⁺均显著高于S组和M组,而CD8+均显著低于S组和M组（P＜0.05）;APSH、APSM和APSL组之间两两相比亦均有统计学差异（P＜0.05）。S组和各APS组PI3K/Akt信号通路蛋白的相对表达均显著低于M组（P＜0.05）;APSH、APSM和APSL组之间两两相比亦均有统计学差异（P＜0.05）。结论：APS可通过调控PI3K/Akt 信号通路和改善免疫功能,发挥对EPC的抑制作用,为临床治疗EPC提供了新的思路和理论依据。     

肝癌肿瘤干细胞的分离鉴定及差异性小分子RNA筛选

旨在分离并鉴定肝癌肿瘤干细胞,并对其异常表达的microRNA进行了初步筛选。首先利用流式细胞术及免疫荧光技术分别在肝癌细胞系及肝癌组织标本中确认了CD90⁺细胞的存在;随后利用免疫磁珠分选技术从肝癌细胞系MHCC97-H中分离出了CD90⁺细胞,化疗药物耐药性实验表明,CD90⁺细胞具有明显的化疗药物耐受能力;两次裸鼠皮下成瘤性实验也表明CD90⁺细胞具有较强的成瘤能力,而CD90^-细胞不具备成瘤能力。上述实验充分说明:CD90⁺细胞具有肝癌肿瘤干细胞特性。因此利用该细胞模型进行了肝癌肿瘤干细胞CD90⁺细胞和非肝癌肿瘤干细胞CD90^-细胞之间差异表达的microRNA的检测,结果表明,CD90⁺细胞与CD90^-细胞之间共有92个microRNAs的表达存在差异性,其中在CD90⁺细胞中高表达的microRNAs有52个;在CD90^-细胞中高表达的microRNAs有40个。     

Hypermethylation of 5'CpG island of p14ARF flanking exon 1 Beta in human colorectal cancers displaying a restricted pattern of p53 overexpression concomitant with increased MDM2 expression

ABSTRACT: BACKGROUND: It has been suggested that inactivation of p14ARF, a tumor suppressor central to regulating p53 protein stability through interaction with the MDM2 oncoprotein, abrogates p53 activity in human tumors retaining the wild-type TP53 gene. Differences in expression of tumor suppressor genes are frequently associated with cancer. We previously reported on a pattern of restricted p53 immunohistochemical overexpression significantly associated with microsatellite instability (MSI), low TP53 mutation frequency, and MDM2 overexpression in colorectal cancers (CRCs). In this study, we investigated whether p14ARF alterations could be a mechanism for disabling the p53 pathway in this subgroup of CRCs. RESULTS: Detailed maps of the alterations in the p14ARF gene were determined in a cohort of 98 CRCs to detect both nucleotide and copy-number changes. Methylation-specific PCR combined with bisulfite sequencing was used to evaluate the prevalence and distribution of p14ARF methylation. p14ARF alterations were then correlated with MSI status, TP53 mutations, and immunohistochemical expression of p53 and MDM2. The frequency of p14ARF mutations was extremely low (1/98; 1%), whereas coexistence of methylated and unmethylated alleles in both tumors and normal colon mucosa was common (91/98; 93%). Only seven of nine tumors (7%) had a distinct pattern of methylation compared with normal colon mucosa. Evaluation of the prevalence and distribution of p14ARF promoter methylation in a region containing 27 CpG sites in 35 patients showed a range of methylated CpG sites in tumors (0 to 25 (95% CI 1 to 13) versus 0 to 17 (95% CI 0 to 2)) in adjacent colon mucosa (P = 0.004). Hypermethylation of the p14ARF promoter was significantly correlated with the restricted p53 overexpression pattern (P = 0.03), and MDM2 overexpression (P = 0.02), independently of MSI phenotype. Although no significant correlation between p14ARF methylation and TP53 mutational status was seen (P = 0.23), methylation involving the proximal CpG sites within the 5' CpG flanking exon 1beta was present more frequently in tumors with restricted p53 overexpression than in those with diffuse p53 overexpression (range of methylated clones 17 to 36% (95% CI 24 to 36%) versus range 0 to 3% (95% CI 0 to 3%), P = 0. 0003). CONCLUSION: p14ARF epigenetic silencing may represent an important deregulating mechanism of the p53- MDM2-p14ARF pathway in CRCs exhibiting a restricted p53 overexpression pattern.     

Apoptosis induced by adenovirus-mediated p14ARF expression in U2OS osteosarcoma cells is associated with increased Fas expression

The INK4A/ARF locus on chromosome 9 is a tumor suppressor gene frequently mutated in human cancers. In order to study the effects of p14ARF expression in tumor cells, we constructed a recombinant adenovirus containing p14ARF cDNA (Adp14ARF). Adp14ARF infection of U2OS osteosarcoma cells which has wild type p53 and mutant p14ARF revealed high levels of p14 (ARF) expression within 24h. In addition, Adp14ARF-mediated expressing of p14 (ARF) was associated with increased levels of p53, p21, and mdm2 protein. Growth inhibition assays following Adp14ARF infection demonstrated that the growth of U2OS cells was inhibited relative to infection with control virus. Furthermore, TUNEL analysis as well as PARP cleavage assays demonstrated that Adp14ARF infection was associated with increased apoptosis in U2OS cell line and that it was associated with Adp14ARF induced overexpression of Fas and Fas-L. Addition of Fas-L neutralizing antibody NOK-1 decreased Adp14-mediated cell death, indicating that p14 (ARF) induction of the Fas pathway is associated with increased apoptosis. The finding that Adp14ARF infection did not induce Fas expression in U2OS/E6 and MCF/E6 cells suggests that wild type p53 expression may be necessary for Adp14ARF-mediated induction of Fas. The observation that overexpression of p53 by Adp53 infection in MCF-7 does not induce increased Fas protein levels nor apoptotic cell death suggests that p53 overexpression is required but not sufficient enough for apoptosis. These studies suggest there are other mechanisms other than induction of p53 in ARF-mediated apoptosis and gene therapy using Adp14ARF may be a promising treatment option for human cancers containing wild type p53 and mutant or deleted p14 expression.     

A novel proapoptotic gene PANO encodes a post-translational modulator of the tumor suppressor p14ARF

The protein p14ARF is a known tumor suppressor protein controlling cell proliferation and survival, which mainly localizes in nucleoli. However, the regulatory mechanisms that govern its activity or expression remain unclear. Here, we report that a novel proapoptotic nucleolar protein, PANO, modulates the expression and activity of p14ARF in HeLa cells. Overexpression of PANO enhances the stability of p14ARF protein by protecting it from degradation, resulting in an increase in p14ARF expression levels. Overexpression of PANO also induces apoptosis under low serum conditions. This effect is dependent on the nucleolar localization of PANO and inhibited by knocking-down p14ARF. Alternatively, PANO siRNA treated cells exhibit a reduction in p14ARF protein levels. In addition, ectopic expression of PANO suppresses the tumorigenicity of HeLa cells in nude mice. These results indicate that PANO is a new apoptosis-inducing gene by modulating the tumor suppressor protein, p14ARF, and may itself be a new candidate tumor suppressor gene.     

Analysis of p53 tumor suppressor pathway genes in chronic lymphocytic leukemia

The p53 tumor suppressor gene plays an important role in preventing tumor development. The p53 protein interacts with other p53 signal pathway members to control cell proliferation. In this study, expression of the p53, Human homolog of murine Double Minute 2 (HDM2), p14Alternating Reading Frame (ARF), Zinc Finger and BTB domain containing 7A (ZBTB7A), and B-Cell Lymphoma 6 (BCL6) genes was quantitatively investigated by real-time polymerase chain reaction (PCR) in the peripheral blood of patients with chronic lymphocytic leukemia (CLL) and healthy controls. Plasma fibronectin levels were determined by enzyme-linked immunosorbent assay. Expression of the p53, p14, and HDM2 genes were significantly higher in the patients. However, ZBTB7A and BCL6 gene expression was not detectable in both groups. A positive correlation between p14ARF and HDM2 expression and a negative correlation between p53 and p14ARF expression was observed. Expression of the p14ARF and HDM2 genes were inversely correlated in the control group. Neither HDM2 nor p14ARF gene expression was correlated with p53 expression. The p53 gene was also analyzed for the presence of mutations. A splice-site mutation was found in a single patient. Our findings indicate that expression of the p53, p14ARF, and HDM2 genes are associated with CLL. Elucidation of the mutual interactions at the protein level warrants further studies.     

Distinct E2F-mediated transcriptional program regulates p14ARF gene expression

The tumor suppressor p14(ARF) gene is induced by ectopically expressed E2F, a positive regulator of the cell cycle. The gene is expressed at low levels in normally growing cells in contrast to high levels in varieties of tumors. How p14(ARF) gene is regulated by E2F in normally growing cells and tumor cells remains obscure. Here we show that regulation of p14(ARF) gene by E2F is distinct from that of classical E2F targets. It is directly mediated by E2F through a novel E2F-responsive element that varies from the typical E2F site. The element responds to E2F activity resulting from ectopic E2F1 expression, inactivation of pRb by adenovirus E1a or shRNA, but not to phosphorylation of pRb by serum stimulation or ectopic cyclin D1/cyclin-dependent kinase-4 expression in normal human fibroblasts. The element has activity in various tumor cells with defective pRb, but not in normally growing cells. These results indicate that the distinct regulation constitutes the basis of p14(ARF) function as a tumor suppressor, discriminating abnormal growth signals caused by defects in pRb function from normal growth signals.