新型microRNA—miR-34在肿瘤中的研究进展

miR-34是一类保守的、非编码miRNA。人类miR-34包括miR-34a、miR-34b和miR-34c等,在多种肿瘤中都呈现非正常表达。miR-34通过被p53激活,抑制E2F3、Bcl-2、c—myc、CDK4、CDK6、Cyclin D1以及Cyclin E2的表达,使肿瘤细胞停滞在G1期,抑制肿瘤细胞的生长,诱导肿瘤细胞凋亡,并通过E2F3、SIRT1与p53形成正反馈环路,不断增强其自身和p53的作用。本文就miR-34的研究进展进行综述。     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation, and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV-hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.Key words: UVB irradiation, p53, DNA damage, DNA damage responses, apoptosis, senescence     

The p53-p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53-p21^WAF1 checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21^WAF1-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53-p21^WAF1 checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G₁ arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53−/− and HCT116-p21−/− cells, indicating that the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G₁ arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53-p21^WAF1 pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on the stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53-p21^WAF1 checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1.     

PLGA-Loaded Gold-Nanoparticles Precipitated with Quercetin Downregulate HDAC-Akt Activities Controlling Proliferation and Activate p53-ROS Crosstalk to Induce Apoptosis in Hepatocarcinoma Cells

Controlled release of medications remains the most convenient way to deliver drugs. In this study, we precipitated gold nanoparticles with quercetin. We loaded gold-quercetin into poly(DL-lactide-co-glycolide) nanoparticles (NQ) and tested the biological activity of NQ on HepG2 hepatocarcinoma cells to acquire the sustained release property. We determined by circular dichroism spectroscopy that NQ effectively caused conformational changes in DNA and modulated different proteins related to epigenetic modifications and c ell cycle control. The mitochondrial membrane potential (MMP), reactive oxygen species (ROS), cell cycle, apoptosis, DNA damage, and caspase 3 activity were analyzed by flow cytometry, and the expression profiles of different anti- and pro-apoptotic as well as epigenetic signals were studied by immunoblotting. A cytotoxicity assay indicated that NQ preferentially killed cancer cells, compared to normal cells. NQ interacted with HepG2 cell DNA and reduced histone deacetylases to control cell proliferation and arrest the cell cycle at the sub-G stage. Activities of cell cycle-related proteins, such as p21^WAF, cdk1, and pAkt, were modulated. NQ induced apoptosis in HepG2 cells by activating p53-ROS crosstalk and induces epigenetic modifications leading to inhibited proliferation and cell cycle arrest.     

microRNAs与TP53基因调控网络研究进展

TP53基因(编码p53蛋白)作为一个重要的抑瘤基因,通过调控一系列信号转导通路广泛参与了多种恶性肿瘤的发生发展,一直是肿瘤分子生物学研究领域的热点.最近的研究发现,microRNAs(miRNAs)参与了TP53的信号通路,它们之间存在着复杂的调控网络.一方面,p53通过调控一些miRNAs的转录及转录后成熟,促进细胞周期阻滞、诱导细胞凋亡和衰老,抑制肿瘤发生.另一方面,许多miRNAs,如miR-25、miR-30d、miR-125b和miR-504等可直接调控p53的表达与活性,参与TP53信号通路的调节,还有一些miRNAs则通过调节p53上下游基因,发挥重要的生物学功能.其中,最具有代表性的是miR-34家族,它们受p53直接调控并参与TP53信号通路,通过靶向抑制多个TP53信号通路关键分子的表达,发挥抑瘤作用.此外,它们还可以通过抑制沉默信息调节子,增强p53的活性,反馈调节TP53信号通路.miRNAs与TP53之间调控网络的研究,是对TP53抑瘤机制的重要补充.     

Primary hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes exhibit distinct erythroid phenotype in vitro

Diamond-Blackfan anemia (DBA) is caused by aberrant ribosomal biogenesis due to ribosomal protein (RP) gene mutations. To develop mechanistic understanding of DBA pathogenesis, we studied CD34⁺ cells from peripheral blood of DBA patients carrying RPL11 and RPS19 ribosomal gene mutations and determined their ability to undergo erythroid differentiation in vitro. RPS19 mutations induced a decrease in proliferation of progenitor cells, but the terminal erythroid differentiation was normal with little or no apoptosis. This phenotype was related to a G₀/G₁ cell cycle arrest associated with activation of the p53 pathway. In marked contrast, RPL11 mutations led to a dramatic decrease in progenitor cell proliferation and a delayed erythroid differentiation with a marked increase in apoptosis and G₀/G₁ cell cycle arrest with activation of p53. Infection of cord blood CD34⁺ cells with specific short hairpin (sh) RNAs against RPS19 or RPL11 recapitulated the two distinct phenotypes in concordance with findings from primary cells. In both cases, the phenotype has been reverted by shRNA p53 knockdown. These results show that p53 pathway activation has an important role in pathogenesis of DBA and can be independent of the RPL11 pathway. These findings shed new insights into the pathogenesis of DBA.     

Magnolol induces apoptosis in osteosarcoma cells via G0/G1 phase arrest and p53-mediated mitochondrial pathway

Osteosarcoma is a highly invasive primary malignancy of bone. Magnolol is biologically active, which shows antitumor effects in a variety of cancer cell lines. However, it has not been elucidated magnolol's effects on human osteosarcoma cells (HOC). This study aimed to determine antitumor activity of magnolol and illustrate the molecular mechanism in HOC. Magnolol showed significant inhibition effect of growth on MG-63 and 143B cells and induced apoptosis and cell cycle arrest at G0/G1. In osteosarcoma cells, magnolol upregulated expressions of proapoptosis proteins and suppressed expressions of antiapoptosis proteins. Additionally, under the pretreatment of pifithrin-a (PFT-a, a p53 inhibitor), the magnolol-induced apoptosis was significantly reversed. The results above indicated that magnolol induces apoptosis in osteosarcoma cells may via G0/G1 phase arrest and p53-mediated mitochondrial pathway.     

p53 is important for the anti-proliferative effect of ibuprofen in colon carcinoma cells

S-ibuprofen which inhibits the cyclooxygenase-1/-2 and R-ibuprofen which shows no COX-inhibition at therapeutic concentrations have anti-carcinogenic effects in human colon cancer cells; however, the molecular mechanisms for these effects are still unknown. Using HCT-116 colon carcinoma cell lines, expressing either the wild-type form of p53 (HCT-116 p53^wt) or being p(HCT-116 p53^−/−), we demonstrated that both induction of a cell cycle block and apoptosis after S- and R-ibuprofen treatment is in part dependent on p53. Also in the in vivo nude mice model HCT-116 p53^−/− xenografts were less sensitive for S- and R-ibuprofen treatment than HCT-116 p53^wt cells. Furthermore, results indicate that induction of apoptosis in HCT-116 p53^wt cells after ibuprofen treatment is in part dependent on a signalling pathway including the neutrophin receptor p75^NTR, p53 and Bax.     

The p53-signaling pathway and colorectal cancer: Interactions between downstream p53 target genes and miRNAs

IntroductionWe examined expression of genes in the p53-signaling pathway. We determine if genes that have significantly different expression in carcinoma tissue compared to normal mucosa also have significantly differentially expressed miRNAs. We utilize a sample of 217 CRC cases.MethodsWe focused on fold change (FC) > 1.50 or <0.67 for genes and miRNAs, that were statistically significant after adjustment for multiple comparisons. We evaluated the linear association between the differential expression of miRNA and mRNA. miRNA:mRNA seed-region matches also were determined.ResultsEleven dysregulated genes were associated with 37 dysregulated miRNAs; all were down-stream from the TP53 gene. MiR-150-5p (HR = 0.82) and miR-196b-5p (HR 0.73) significantly reduced the likelihood of dying from CRC when miRNA expression increased in rectal tumors.ConclusionsOur data suggest that activation of p53 from cellular stress, could target downstream genes that in turn could influence cell cycle arrest, apoptosis, and angiogenesis through mRNA:miRNA interactions.     

p53‐mediated regulation of cell cycle progression: Pronounced impact of cellular microenvironment

Data on the biological effects of some overexpressed oncogenes and their cooperation with cellular factors are, at least partially, contradictory. There are reports on the strong pro‐apoptotic action of temperature‐sensitive (ts) p53^135val in transformed cells at permissive temperature. However, in our experience very high levels of p53^135val induce in transformed rat cells at permissive temperature cell cycle arrest but not apoptosis. Comparison of the experimental protocols reveals that cells used for transfection strongly differ. Therefore, we decided to explore the impact of primary cells used for generation of cell clones on the biological effects evoked by p53 and c‐Ha‐Ras. In the present study, we used primary rat cells (RECs) isolated from rat embryos of different age: at 13.5 gd (y) and 15.5 gd (o). We immortalized rat cells using ts p53^135val mutant and additionally generated transformed cells after co‐transfection with oncogenic Ras. The RECs were transfected with a constitutively activated Ha‐Ras protein, a mutation that is found in a wide variety of human tumors. The ts p53^135Val mutant, switching between wild‐type (wt) and mutant conformation, offers the possibility to study the escape from p53‐mediated cell cycle control in a model of malignant transformation in cells with the same genetic background. Surprisingly, the kinetics of cell proliferation at non‐permissive temperature and that of cell cycle arrest at 32°C strongly differed between cell clones established from yRECs and oRECs, thereby indicating that overexpression of genes such as ts p53^135Val mutant and oncogenic‐Ha‐Ras does not fully override the intrinsic cellular program. J. Cell. Physiol. 219: 459–469, 2009. © 2009 Wiley‐Liss, Inc.     

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.