Gadd45a的表达调控与功能

哺乳动物细胞对于遗传毒性的刺激会产生一系列应答,如细胞周期阻滞,DNA修复和细胞凋亡等。Gadd45a在DNA损伤诱导的细胞应答中发挥重要作用。细胞内外环境的多种因素在转录水平、转录后水平、翻译后水平等多个层次对Gadd45a进行精确调节。Gadd45a通过与Cdc2相互作用调控细胞周期G1-M检测点,直接抑制Aurora—A激酶参与中心体稳定性的调节,通过G1-S期调控参与维持基因组的稳定性。Gadd45a参与p38／JNK、MAPK、线粒体介导的凋亡途径和NF—κB介导的生存通路调控。     

Gadd45α在干细胞DNA损伤中的作用

基因组完整性对于细胞和组织功能至关重要,这种稳态会不断地受到内源性和外源性应激刺激的影响.干细胞对这些应激刺激十分敏感,其DNA会发生不同程度的损伤,诱导干细胞内固有的DNA修复机制.组织特异性干细胞是局部环境中的多能群体,在其整个生命过程中负责维持组织或者系统的完整性.组织特异性干细胞在受到应激刺激之后,能通过某些反...     

Gadd45a基因对小鼠造血干细胞功能的影响

目的 Gadd45a基因对小鼠造血干细胞功能的影响。方法流式细胞仪分选小鼠骨髓造血干细胞、体外单克隆培养,竞争性骨髓移植,放射线照射观察生存曲线。结果 Gadd45a基因缺失的小鼠造血干细胞克隆形成能力增强,短期造血重建能力无差异,8.5Gy放射线照射后生存情况无差异。结论 Gadd45a基因对小鼠造血干细胞功能起重要作用。     

Gadd45g诱导小鼠胚胎干细胞向中内胚层分化

为探究生长阻滞和DNA损伤诱导蛋白45γ(Growth arrest and DNA-damage-inducible protein GADD45 gamma,Gadd45g)对小鼠胚胎干细胞(mouse embryonic stem cells,m ESCs)在体外培养条件下自我更新状态的影响,通过设计并构建含有Gadd45g基因的重组质粒,将其导入m ESCs内,过表达目标基因;在含有白血病抑制因子(LIF)的血清培养条件下,通过细胞计数、碱性磷酸酶染色、qRT-PCR以及免疫荧光等实验手段检测m ESCs的生长情况。结果显示,与对照组相比,过表达Gadd45g基因后,m ESCs的生长速度减缓,碱性磷酸酶活性降低,且中内胚层标志基因的表达水平显著上升。进一步研究发现,在添加LIF的有血清或2i无血清培养体系中,过表达Gadd45g均可以降低细胞内STAT3蛋白的磷酸化水平,由此推断上调Gadd45g的表达会抑制STAT3的活性,从而推动m ESCs向中内胚层分化。研究结果扩大了目前人们对于ESCs分化机制的理解,有利于胚胎干细胞未来的基础研究与安全应用。     

牛体细胞中Gadd45a的表达与DNA甲基化修饰

目的 检测成年牛心脏、肾脏、肝脏及睾丸组织中Gadd45a基因表达情况及DNA甲基化状态,并说明二者之间的关系.结果 牛Gadd45a基因在心脏中没有表达,而在其他3种组织中有表达且略有差异.选定该基因调控区中的7个CpG位点,利用甲基化特异性PCR检测CpG位点的DNA甲基化状态,结果心脏中的甲基化程度明显高于其他3种组织.结论 DNA甲基化对牛Gadd45a基因的组织特异性表达有一定影响.     

Solution Structure of Human Growth Arrest and DNA Damage 45α (Gadd45α) and Its Interactions with Proliferating Cell Nuclear Antigen (PCNA) and Aurora A Kinase

Gadd45α is a nuclear protein encoded by a DNA damage-inducible gene. Through its interactions with other proteins, Gadd45α participates in the regulation of DNA repair, cell cycle, cell proliferation, and apoptosis. The NMR structure of human Gadd45α has been determined and shows an α/β fold with two long disordered and flexible regions at the N terminus and one of the loops. Human Gadd45α is predominantly monomeric in solution but exists in equilibrium with dimers and other oligomers whose population increases with protein concentration. NMR analysis shows that Aurora A interacts through its N-terminal domain with a region of human Gadd45α encompassing the site of dimerization, suggesting that the oligomerization of Gadd45α could be a regulatory mechanism to modulate its interactions with Aurora A, and possibly with other proteins too. However, Gadd45α appears to interact only weakly with PCNA through its flexible loop, in contrast with previous and contradictory reports.     

Gadd45a在抑制细胞转化和肿瘤恶性进展中的作用

Gadd45a,一个受p53和BRCA1调节的生长阻滞和DNA损伤基因,在抑制细胞转化和肿瘤恶性进展中扮演重要的角色.Gadd45a可以通过抑制细胞生长以及促进DNA损伤修复等间接或者直接方式维持基因组稳定性,从而抑制细胞转化和肿瘤的恶性进展.此外,Gadd45a还可通过对一些信号传导通路的调节,参与肿瘤发生发展的抑制.     

Gene expression profile and overexpression of apoptosis-related genes (NGFI-B and Gadd 45 γ) in early phase of Thy-1 nephritis model

Mesangioproliferative glomerulonephritis (MPGN) is a disease of high incidence in humans. Rat Thy-1 nephritis (Thy-1 N), namely, anti-thymocyte serum (ATS)-induced nephritis, is considered to be an animal model for studying MPGN. Although previous studies have demonstrated that glomerular mesangial cell (GMCs) injury might be a feature of Thy-1 N, the mechanism of the disease (i.e., GMC apoptosis) remains unclear. We have examined the pathologic changes of GMCs and the gene expression profile of renal tissues in Thy-1 N. The pathologic changes of Thy-1 N include three phages: GMC apoptosis (40 min), necrosis (2 h), and proliferation (5 days). Many TUNEL-positive cells are found 40 min after administration of ATS. Concomitantly, 341 genes are up-regulated, whereas 392 genes are down-regulated, as shown by microarrays analysis. The mRNA and protein of two of the up-regulated genes (nerve growth factor induced protein I-B, NGFI-B; growth arrest- and DNA-damage-inducible protein 45 gamma, Gadd 45 γ) in the GMC apoptotic phase of Thy-1 N are markedly elevated, as observed by real-time polymerase chain reaction and immunohistochemistry. Our data indicate that pathologic changes of Thy-1 N are involved in the abnormal gene profile. The overexpression of the NGFI-B and Gadd 45 γ genes may be associated with GMC apoptosis of Thy-1 N.This work was supported by grants (no. 30571728, 30471615, and no. 03KJA310074) from the National Natural Science Foundations of China and Jiangsu Province.     

Gadd45α as an upstream signaling molecule of p38 MAPK triggers oxidative stress-induced sFlt-1 and sEng upregulation in preeclampsia

Preeclampsia (PE) is known to be associated with increased circulating levels of anti-angiogenic factors, such as soluble fms-related tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng). However, the way that placental oxidative stress results in the elevation of these two factors remains enigmatic. We have observed the overexpression of growth arrest and DNA damage-inducible 45 alpha (Gadd45α) and excessive activation of p38 mitogen-activated protein kinase (MAPK) in preeclamptic placentas compared with normotensive controls, together with increased levels of sFlt-1 and sEng in maternal sera in patients with PE. Moreover, Gadd45α knockdown or p38 inhibition provides protective effects in hypoxia/reoxygenation (H/R)-exposed human umbilical vein endothelial cells (HUVECs) by suppressing oxidative stress, inhibiting apoptosis, and promoting their potential for in vitro angiogenesis. A regulatory signaling pathway in which H/R intervention causes the induction of Gadd45α leading to p38 activation and ultimately an increase in sFlt-1 and sEng secretion in HUVECs has concurrently been established. Our study opens up a promising new avenue of investigation for increasing the understanding of the origin of sFlt-1 and sEng in PE and provides novel therapeutic targets for pregnancy complications arising from placental endothelial dysfunction.     

p53转录活化的p21和Gadd45在细胞DNA损伤监视中的作用

p2 1,Gadd45均是受抑癌基因p5 3转录调控的分子 ,当电离辐射引起细胞DNA损伤时 ,p2 1,Gadd45基因的转录以p5 3依赖的方式增强 .为了深入探讨p2 1,Gadd45在DNA损伤监视中的作用 ,构建了稳定表达p2 1及Gadd45反义RNA的p2 1^as/MCF 7及Gadd45 ^as/MCF 7细胞系 .对辐射引起这些细胞G₁期阻滞、DNA损伤修复和细胞凋亡的发生进行了研究 ,发现Gadd45同p2 1一样 ,当其表达受阻后辐射引起的细胞G₁期阻滞明显减弱 ,宿主细胞对辐射损伤的报告基因的修复减弱 ,还观察到辐射引起的p2 1表达受阻的细胞调亡的增加 ,结果表明p2 1,Gadd45通过参与多种辐射细胞学反应发挥DNA损伤监视作用 .     

Tissue and serum α2-3- and α2-6-linkage specific sialylation changes in oral carcinogenesis

Increased sialylation of cell surface glycoconjugates is among the key molecular changes associated with malignant transformation and cancer progression. We investigated significance of linkage-specific sialylation changes in oral carcinogenesis. Tissue and serum levels of total sialic acid (TSA), linkage-specific sialyltransferases (ST) and sialoproteins were analyzed from patients with oral precancerous conditions (OPC) and oral cancer as well as the post-treatment follow-up blood samples of oral cancer patients. TSA levels were measured using a spectrophotometric method. The linkage-specific lectins, Sambusus nigra (SNA) and Maackia amurensis (MAM) detects α2-6- and α2-3-linked sialic acid, respectively, were used to analyze ST activity and sialoproteins. Malignant tissues showed significantly higher levels of TSA, reactivity of SNA and MAM, and α2,3-ST activity compared to the adjacent normal tissues. α2,6-ST was also higher in malignant tissues. Similarly, the marker levels were higher in precancerous tissues than their adjacent normal tissues. Serum levels of TSA, TSA/ total proteins, α2-6-sialoproteins and α2,6-ST were markedly increased in untreated oral cancer patients compared to the controls and OPC as well as responder (CR) patients. Serum levels of the markers were higher or comparable between untreated oral cancer patients and non-responders (NR). Serum levels of α2-3-sialylation were elevated in non-responders compared with the responders. Further, the observed sialylation changes in tissue and serum were found to be associated with various clinicopathological features and disease progression. Thus, the data suggest potential utility of sialylation markers in early detection, prognostication and treatment monitoring of oral cancer.     

Surface α2-3- and α2-6-sialylation of human monocytes and derived dendritic cells and its influence on endocytosis

Several glycoconjugates are involved in the immune response. Sialic acid is frequently the glycan terminal sugar and it may modulate immune interactions. Dendritic cells (DCs) are antigen-presenting cells with high endocytic capacity and a central role in immune regulation. On this basis, DCs derived from monocytes (mo-DC) are utilised in immunotherapy, though many features are ignored and their use is still limited. We analyzed the surface sialylated glycans expressed during human mo-DC generation. This was monitored by lectin binding and analysis of sialyltransferases (ST) at the mRNA level and by specific enzymatic assays. We showed that α2-3-sialylated O-glycans and α2-6- and α2-3-sialylated N-glycans are present in monocytes and their expression increases during mo-DC differentiation. Three main ST genes are committed with this rearrangement: ST6Gal1 is specifically involved in the augmented α2-6-sialylated N-glycans; ST3Gal1 contributes for the α2-3-sialylation of O-glycans, particularly T antigens; and ST3Gal4 may contribute for the increased α2-3-sialylated N-glycans. Upon mo-DC maturation, ST6Gal1 and ST3Gal4 are downregulated and ST3Gal1 is altered in a stimulus-dependent manner. We also observed that removing surface sialic acid of immature mo-DC by neuraminidase significantly decreased its endocytic capacity, while it increased in monocytes. Our results indicate the STs expression modulates the increased expression of surface sialylated structures during mo-DC generation, which is probably related with changes in cell mechanisms. The ST downregulation after mo-DC maturation probably results in a decreased sialylation or sialylated glycoconjugates involved in the endocytosis, contributing to the downregulation of one or more antigen-uptake mechanisms specific of mo-DC.     

Chemoenzymatic synthesis of C8-modified sialic acids and related α2-3- and α2-6-linked sialosides

Naturally occurring 8-O-methylated sialic acids, including 8-O-methyl-N-acetylneuraminic acid and 8-O-methyl-N-glycolylneuraminic acid, along with 8-O-methyl-2-keto-3-deoxy-d-glycero-d-galacto-nonulosonic acid (Kdn8Me) and 8-deoxy-Kdn were synthesized from corresponding 5-O-modified six-carbon monosaccharides and pyruvate using a sialic acid aldolase cloned from Pasteurella multocida strain P-1059 (PmNanA). In addition, α2-3- and α2-6-linked sialyltrisaccharides containing Neu5Ac8Me and Kdn8Deoxy were also synthesized using a one-pot multienzyme approach. The strategy reported here provides an efficient approach to produce glycans containing various C8-modified sialic acids for biological evaluations.     

Synthesis and Properties of 2′4′- and 3′-5′-Linked Ribodinucleoside Monophosphates Containing 2-Aminoadenosine and Uridine

Abstract

Self complementary diribonucleoside monophosphates containing 2-aminoadenosine (n²A) and uridine (U) residues, (2′-5′) n²ApU (1), (3′-5′) n²ApU (2), (2′-5′) Upn²A (3) and (3′-5′) Upn²A (4), were synthesized by condensation of suitably protected nucleoside and nucleotide units using dicyclohexylcarbodiimide (DCC). The dimers, (3) and (41, were also obtained from uridine 2′,3′-cyclic phosphate and unprotected 2-aminoadenosine using 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl) as the condensing agent. The conformational properties of these dimers were examined by UV, CD and NMR spectroscopy. The results reveal that the 2′-5′ isomers take a stacked conformation, which contains a larger base-base overlap and is more stable against thermal perturbation with respect to the 3′-5′ isomers. The n²ApU isomers have more stacked structure than the Upn²A isomers.     

Synthesis of 7-(tetrahydropyran-2-yl)- and 7-(4-acetoxytetrahydropyran-2-yl)-ε-rhodomycinone and 7-(tetrahydropyran-2-yl)-ε-pyrromycinone

The ¹³C.n.m.r spectra of water-soluble and -insoluble glucans synthesized by enzymes isolated from six strains of Streptococcus mutans are interpreted. The glucans are shown to be composed primarily of α(1→3)- and α-(1→6)-linked glucosyl residues, and the relative abundance of each linkage is estimated from peak areas. Treatment of water-insoluble glucans with dextranase is found to result in water-soluble and -insoluble products, the former enriched in α-(1→6)-linkages and the latter in α-(1→3)-linkages. The structural conclusions arrived at by ¹³C-n.m.r. spectroscopy are consistent with data from methylation analysis and ¹H-n.m.r. spectroscopy.     

HDAC inhibitors induce apoptosis but not cellular senescence in Gadd45α-deficient E1A+Ras cells

HDAC inhibitors (HDIs) induce irreversible cell cycle arrest and senescence in E1A+Ras expressing cells. Furthermore, HDIs activate Gadd45α/NF-κB signaling pathway to suppress apoptosis thereby promoting the cell survival. Here, to clarify the role of Gadd45α in realization of the antiapoptotic program, we compared wild-type E1A+Ras cells and the cells with knockout of gadd45α gene (Gadd45α−/− cells). As in Gadd45α-expressing E1A+Ras cells, HDIs induce irreversible cell cycle arrest in Gadd45α−/− cells, but the arrested cells do not senesce and eventually die due to activation of the apoptotic death program. These data suggest that the expression of Gadd45α is involved in maintaining the balance of pro- and anti-apoptotic stimuli, while lack or loss of Gadd45 directs the cells to apoptosis after HDIs treatment. Appropriately Gadd45α-deficient cells demonstrate a higher level of pro-apoptotic signals, whereas the anti-apoptotic program is suppressed. The elevated apoptotic background of Gadd45α−/− cells is accompanied by higher levels of Ser15-phosphorylated p53 and p21/Waf1 proteins that additionally commit the cells to HDIs-induced apoptosis. Additionally, loss of Gadd45α protein activates the DDR signaling pathway as demonstrated by nuclear pATM staining, accumulation of γH2AX foci and an increase of single-strand DNA breaks. Thus, in wild-type E1A+Ras cells the p53-dependent expression of Gadd45α is necessary not only for DNA repair and HDI-induced cellular senescence, but also to withstand to apoptosis after DNA damage and stress. Therefore the use of HDIs in combination with agents that block Gadd45α function may have promise for cancer therapy.     

Methylation mediated Gadd45β enhanced the chemosensitivity of hepatocellular carcinoma by inhibiting the stemness of liver cancer cells

Background

Defects of the growth arrest DNA damage-inducible gene 45β (Gadd45β) play an important role in the progression of tumor and confer resistance to chemotherapy. However, the role of Gadd45β in the apoptosis of hepatocellular carcinoma is still not clear. Purpose of this study was to explore the effect of Gadd45β on the apoptosis of liver cancer cells, and the possible mechanism was examined.

Result

In this study, we first confirmed the decreased expression of Gadd45β in human liver cancer tissues and human liver cancer cell lines, when compared to the peri-tumor liver tissue and normal liver cells. And, it was found that Gadd45β could inhibit the stemness of liver cancer cells, enhancing the apoptosis of cancer cells induced by chemotherapy. Furthermore, the results showed that HCC tissues and cell lines showed a higher methylation status in Gadd45β promoter than that in peri-tumor tissues and normal liver cells. Methylation was then reversed by pretreatment of SMMC-7721 and Hep-3B with 5-azacytidine which is the DNA methyltransferase inhibitor. And the 5-azacytidine decreased the stemness of SMMC-7721 and Hep-3B, enhanced the sensitivity of SMMC-7721 and Hep-3B to cisplatin.

Conclusions

Methylation mediated Gadd45β expression inhibited the stemness of liver cancer cells, promoting the chemotherapy-induced apoptosis. Thus Gadd45β may be the potential target for enhancing the chemosensitivity of human hepatocellular carcinoma.

     

Stereocontrolled preparation of C-2-deoxy-α- and -β-d-glucopyranosyl compounds from tributyl-(2-deoxy-α- and -β-d-glucopyranosyl)stannanes

tributylstannyllithium treatment of 3,4,6-tri-O-benzyl-2-deoxy-α-d-arabino-hexopyranosyl chloride (2) provided selectively tributyl (3,4,6-tri-O-benzyl-2-deoxy-β-d-arabino-hexopyranosyl)stannane (3) in 85% yield. Isomeric tributyl (3,4,6-tri-O-benzyl-2-deoxy-α-d-arabino-hexopyranosyl)stannane (6) could be prepared in 70% yield by reductive lithiation of 2 and reaction with tributyltin chloride. Tin—lithium exchange reaction, performed on 3 and 6 with butyllithium in oxolane at −78°, generated the corresponding, configurationally stable 2-deoxy-β- and -α-d-hexopyranosyllithium compounds which reacted with electrophilic compounds with retention of configuration. Addition to these glycosyllithium reagents to prochiral carbonyl compounds gave variable degrees of facial selectivity. A significant diastereofacial discrimination (10:1) was observed by condensation of 3,4,6-tri-O-benzyl-2-deoxy-α-d-arabino-hexopyranosyllithium reagent with hexanal and isobutyraldehyde. The structure of all C-glycopyranosyl compounds obtained was established by ¹H-n.m.r. spectroscopy.