The RASSF6 Tumor Suppressor Protein Regulates Apoptosis and the Cell Cycle via MDM2 Protein and p53 Protein

Ras association domain family (RASSF) 6 is a member of the C-terminal RASSF proteins such as RASSF1A and RASSF3. RASSF6 is involved in apoptosis in various cells under miscellaneous conditions, but it remains to be clarified how RASSF6 exerts tumor-suppressive roles. We reported previously that RASSF3 facilitates the degradation of MDM2, a major E3 ligase of p53, and stabilizes p53 to function as a tumor suppressor. In this study, we demonstrate that RASSF6 overexpression induces G₁/S arrest in p53-positive cells. Its depletion prevents UV- and VP-16-induced apoptosis and G₁/S arrest in HCT116 and U2OS cells. RASSF6-induced apoptosis partially depends on p53. RASSF6 binds MDM2 and facilitates its ubiquitination. RASSF6 depletion blocks the increase of p53 in response to UV exposure and up-regulation of p53 target genes. RASSF6 depletion delays DNA repair in UV- and VP-16-treated cells and increases polyploid cells after VP-16 treatment. These findings indicate that RASSF6 stabilizes p53, regulates apoptosis and the cell cycle, and functions as a tumor suppressor. Together with the previous reports regarding RASSF1A and RASSF3, the stabilization of p53 may be the common function of the C-terminal RASSF proteins.     

Oncogenic MicroRNA-155 Down-regulates Tumor Suppressor CDC73 and Promotes Oral Squamous Cell Carcinoma Cell Proliferation: IMPLICATIONS FOR CANCER THERAPEUTICS*

The CDC73 gene is mutationally inactivated in hereditary and sporadic parathyroid tumors. It negatively regulates β-catenin, cyclin D1, and c-MYC. Down-regulation of CDC73 has been reported in breast, renal, and gastric carcinomas. However, the reports regarding the role of CDC73 in oral squamous cell carcinoma (OSCC) are lacking. In this study we show that CDC73 is down-regulated in a majority of OSCC samples. We further show that oncogenic microRNA-155 (miR-155) negatively regulates CDC73 expression. Our experiments show that the dramatic up-regulation of miR-155 is an exclusive mechanism for down-regulation of CDC73 in a panel of human cell lines and a subset of OSCC patient samples in the absence of loss of heterozygosity, mutations, and promoter methylation. Ectopic expression of miR-155 in HEK293 cells dramatically reduced CDC73 levels, enhanced cell viability, and decreased apoptosis. Conversely, the delivery of a miR-155 antagonist (antagomir-155) to KB cells overexpressing miR-155 resulted in increased CDC73 levels, decreased cell viability, increased apoptosis, and marked regression of xenografts in nude mice. Cotransfection of miR-155 with CDC73 in HEK293 cells abrogated its pro-oncogenic effect. Reduced cell proliferation and increased apoptosis of KB cells were dependent on the presence or absence of the 3′-UTR in CDC73. In summary, knockdown of CDC73 expression due to overexpression of miR-155 not only adds a novelty to the list of mechanisms responsible for its down-regulation in different tumors, but the restoration of CDC73 levels by the use of antagomir-155 may also have an important role in therapeutic intervention of cancers, including OSCC.     

Tumor suppressor protein Pdcd4 inhibits translation of p53 mRNA

The tumor suppressor protein Pdcd4 is thought to suppress translation of mRNAs containing structured 5'-UTRs by interacting with translation initiation factor eIF4A and inhibiting its helicase activity. However, natural target mRNAs regulated by Pdcd4 so far are mostly unknown. Here, we identified p53 mRNA as a translational target of Pdcd4. We found that Pdcd4 is associated with p53 mRNA and suppresses its translation. The inhibitory effect of Pdcd4 on the translation of p53 mRNA depends on the ability of Pdcd4 to interact with eIF4A and is mediated by the 5'-UTR of p53 mRNA, which is able to form a stable stem-loop structure. We show that treatment of cells with DNA-damaging agents decreases the expression of Pdcd4. This suggests that translational suppression by Pdcd4 plays a role in maintaining a low level of p53 in unstressed cells and that this suppression is abrogated due to low levels of Pdcd4 after DNA damage. Overall, our work demonstrates for the first time that Pdcd4 is directly involved in translational suppression of a natural mRNA with a 5'-structured UTR and provides novel insight into the translational control of p53 expression.     

MicroRNA 218 Acts as a Tumor Suppressor by Targeting Multiple Cancer Phenotype-associated Genes in Medulloblastoma

Aberrant expression of microRNAs has been implicated in many cancers. We recently demonstrated differential expression of several microRNAs in medulloblastoma. In this study, the regulation and function of microRNA 218 (miR-218), which is significantly underexpressed in medulloblastoma, was evaluated. Re-expression of miR-218 resulted in a significant decrease in medulloblastoma cell growth, cell colony formation, cell migration, invasion, and tumor sphere size. We used C17.2 neural stem cells as a model to show that increased miR-218 expression results in increased cell differentiation and also decreased malignant transformation when transfected with the oncogene REST. These results suggest that miR-218 acts as a tumor suppressor in medulloblastoma. MicroRNAs function by down-regulating translation of target mRNAs. Targets are determined by imperfect base pairing of the microRNA to the 3′-UTR of the mRNA. To comprehensively identify actual miR-218 targets, medulloblastoma cells overexpressing miR-218 and control cells were subjected to high throughput sequencing of RNA isolated by cross-linking immunoprecipitation, a technique that identifies the mRNAs bound to the RNA-induced silencing complex component protein Argonaute 2. High throughput sequencing of mRNAs identified 618 genes as targets of miR-218 and included both previously validated targets and many targets not predicted computationally. Additional work further confirmed CDK6, RICTOR, and CTSB (cathepsin B) as targets of miR-218 and examined the functional role of one of these targets, CDK6, in medulloblastoma.     

58-kDa microspherule protein (MSP58) is novel Brahma-related gene 1 (BRG1)-associated protein that modulates p53/p21 senescence pathway

The nucleolar 58-kDa microspherule protein (MSP58) protein is a candidate oncogene implicated in modulating cellular proliferation and malignant transformation. In this study, we show that knocking down MSP58 expression caused aneuploidy and led to apoptosis, whereas ectopic expression of MSP58 regulated cell proliferation in a context-dependent manner. Specifically, ectopic expression of MSP58 in normal human IMR90 and Hs68 diploid fibroblasts, the H184B5F5/M10 mammary epithelial cell line, HT1080 fibrosarcoma cells, primary mouse embryonic fibroblasts, and immortalized NIH3T3 fibroblasts resulted in induction of premature senescence, an enlarged and flattened cellular morphology, and increased senescence-associated β-galactosidase activity. MSP58-driven senescence was strictly dependent on the presence of functional p53 as revealed by the fact that normal cells with p53 knockdown by specific shRNA or cells with a mutated or functionally impaired p53 pathway were effective in bypassing MSP58-induced senescence. At least two senescence mechanisms are induced by MSP58. First, MSP58 activates the DNA damage response and p53/p21 signaling pathways. Second, MSP58, p53, and the SWI/SNF chromatin-remodeling subunit Brahma-related gene 1 (BRG1) form a ternary complex on the p21 promoter and collaborate to activate p21. Additionally, MSP58 protein levels increased in cells undergoing replicative senescence and stress-induced senescence. Notably, the results of analyzing expression levels of MSP58 between tumors and matched normal tissues showed significant changes (both up- and down-regulation) in its expression in various types of tumors. Our findings highlight new aspects of MSP58 in modulating cellular senescence and suggest that MSP58 has both oncogenic and tumor-suppressive properties.     

肿瘤抑制因子p53功能及其抗病毒作用研究进展

肿瘤抑制因子p53 作为基因组的守护者,能通过细胞周期调控和促进细胞凋亡而阻止癌细胞及机体肿瘤的发生,p53还能参与DNA损伤修复、调节机体代谢及调节繁殖生育等功能。除此以外,近年来研究发现,p53能通过促进病毒感染的细胞凋亡而起到抗病毒作用以及p53受IFN的调控和p53作为转录调控因子还能直接转录激活IRF9、IRF5、ISG15和TLR3等抗病毒基因,从而确定了p53在抗病毒反应中起到重要作用。这表明p53可能参与先天性免疫、获得性免疫及炎症反应而起到抗病毒的作用。     

erbB-2表达抑制与EGF对p21、p53基因转录活性的促进

原癌基因ｅｒｂＢ－２的异常表达存在于人类多种肿瘤中,与肿瘤的发生、发展密切相关［１］．我们曾构建了反义ｅｒｂＢ－２逆转录病毒重组载体,将其转染存在该基因异常表达的人胃癌细胞系ＢＧＣ－８２３,达到了特异抑制ｅｒｂＢ－２表达、抑制瘤细胞恶性增殖并部分阻断...     

p53蛋白多肽片段在大肠杆菌中的表达

在所有研究过的人体肿瘤组织或细胞中,ｐ５３似乎是突变频率最高的一个基因,研究和检测ｐ５３基因及其编码产物的变化将具有重要的意义。我们将野生型ｐ５３基因编码区３’端７０３ｂｐ的ｃＤ－ＮＡ片段插入到大肠杆菌表达载体ｐＢＶ２２０中,得到了一个重组体表达质粒ｐＲＲ３３,经热诱导表达,用ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ印迹法证实ｐ５３蛋白多肽在大肠杆菌中得到了表达,它不仅可用于抗ｐ５３蛋白抗体的制备,而且也可用于对ｐ５３蛋白羧基端多肽功能的研究。