Down-regulation of the expression of beta1,4-galactosyltransferase V promotes integrin beta1 maturation

In previous study, we have shown that beta1,4-galactosyltransferase V (GalT V) functions as a positive growth regulator in glioma. Here, we reported that down-regulation of the expression of GalT V in SHG44 cells by transfection with antisense cDNA specifically up-regulated the expression of cell surface integrin beta1 without the change of its mRNA, and with integrin beta1 125 kDa mature form increased and 105 kDa precursor form decreased. It is well known that the N-glycans of integrins modulate the location and functions of integrins. The SHG44 cells transfected with antisense cDNA of GalT V demonstrated decreased Golgi localization of integrin beta1, strengthened the interaction between integrin alpha5 and beta1 subunit, and enhanced the adhesion ability to fibronectin and the level of focal adhesion kinase phosphorylation. Our results suggested that the down-regulation of the expression of GalT V could promote the expression of cell surface integrin beta1 and subsequently inhibit glioma malignant phenotype.     

Beta1,4-galactosyltransferase V functions as a positive growth regulator in glioma

beta1,4-galactosyltransferase V (GalT V; EC 2.4.1.38) can effectively galactosylate the GlcNAcbeta1-->6Man arm of the highly branched N-glycans that are characteristic of glioma. Previously, we have reported that the expression of GalT V is increased in the process of glioma. However, currently little is known about the role of GalT V in this process. In this study, the ectopic expression of GalT V could promote the invasion and survival of glioma cells and transformed astrocytes. Furthermore, decreasing the expression of GalT V in glioma cells promoted apoptosis, inhibited the invasion and migration and the ability of tumor formation in vivo, and reduced the activation of AKT. In addition, the activity of GalT V promoter could be induced by epidermal growth factor, dominant active Ras, ERK1, JNK1, and constitutively active AKT. Taken together, our results suggest that GalT V functioned as a novel glioma growth activator and might represent a novel target in glioma therapy.     

β1,4-Galactosyltransferase V regulates self-renewal of glioma-initiating cell

Glioma results from unregulated expansion of a self-renewing glioma-initiating cell population. The regulatory pathways which are essential for sustaining the self-renewal of glioma-initiating cells remain largely unknown. Cell surface N-linked oligosaccharides play functional roles in determining cell fate and are associated with glioma malignancy. Previously, we have reported that β1,4-galactosyltransferase V (β1,4GalT V) effectively galactosylates the GlcNAcβ1→6Man arm of the highly branched N-glycans and positively regulates glioma cell growth. Here, we show that decreasing the expression of β1,4GalT V by RNA interference in glioma cells attenuated the formation of polylactosamine and inhibited the ability of tumor formation in vivo. Down-regulation of β1,4GalT V depleted CD133-positive cells in glioma xenograft, and inhibited the self-renewal capacity and the tumorigenic potential of glioma-initiating cells. These data reveal a critical role of β1,4GalT V in the self-renewal and tumorigenicity of glioma-initiating cells, and indicate that manipulating β1,4GalT V expression may have therapeutic potential for the treatment of malignant glioma.     

beta榄香烯对人脑胶质瘤SHG44 细胞增殖及凋亡的影响

目的：探讨中药提取物beta- 榄香烯对胶质瘤SHG44 细胞增殖抑制作用及对Bax 和Bcl-2 蛋白表达的影响,并进一步探讨发 生的机制。方法：用不同浓度的beta-榄香烯对体外培养的SHG44 细胞进行干预,分别采用MTT、流式细胞仪检测法,观察beta-榄香 烯对SHG44细胞增殖的抑制和凋亡诱导作用,并通过Western blot检测凋亡相关蛋白Bax 与Bcl-2 蛋白表达情况。结果：经beta- 榄香烯处理SHG44细胞后,MTT 结果其发现SHG44细胞生长受药物浓度和时间的影响,细胞生长明显被抑制,且（P<0.05）,流 式细胞术显示,茁- 榄香烯诱导SHG44细胞后,细胞凋亡指数伴随药物作用时间的延长凋亡显著增加;Western blot 结果发现,beta- 榄香烯对SHG44 细胞的诱导后,使促凋亡蛋白Bax 和抑凋亡蛋白Bcl-2 与对照组相比发生了显著改变,且实验组Bax 蛋白表达 明显高于对照组,而抑凋亡蛋白Bcl-2 的表达伴随beta- 榄香烯的作用时间的增加,表达也逐渐减少。结论：beta- 榄香烯能显著抑制胶 质瘤SHG44 细胞的增殖,促进其凋亡;其机制可能与调控Bcl-2 和Bax表达有关。     

Expression of β‐1,4‐galactosyltransferase I in rat Schwann cells

Glycosylation is one of the most important post‐translational modifications. It is clear that the single step of β‐1,4‐galactosylation is performed by a family of β‐1,4‐galactosyltransferases (β‐1,4‐GalTs), and that each member of this family may play a distinct role in different tissues and cells. In the present study, real‐time PCR revealed that the β‐1,4‐GalT I mRNA reached peaks at 2 weeks after sciatic nerve crush and 3 days after sciatic nerve transection. Combined in situ hybridization for β‐1,4‐GalT I mRNA and immunohistochemistry for S100 showed that β‐1,4‐GalT I mRNAs were mainly located in Schwann cells after sciatic nerve injury. In conclusion, β‐1,4‐GalT I might play important roles in Schwann cells during the regeneration and degeneration of the injured sciatic nerve. In other pathology, such as inflammation, we found that LPS administration affected β‐1,4‐GalT I mRNA expression in sciatic nerve in a time‐ and dose‐dependent manner, and β‐1,4‐GalT I mRNA is expressed mainly in Schwann cells. These results indicated that β‐1,4‐GalT I plays an important role in the inflammation reaction induced by intraperitoneal injection of LPS. Similarly, we found that β‐1,4‐GalT I in Schwann cells in vitro was affected in a time‐ and concentration‐dependent manner in response to LPS stimulation. All these results suggest that β‐1,4‐GalT I play an important role in Schwann cells in vivo and vitro during pathology. In addition, β‐1,4‐GalT I production was drastically suppressed by U0126 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (SAPK/JNK inhibitor), which indicated that Schwann cells which regulated β‐1,4‐GalT I expression after LPS stimulation were via ERK, SAPK/JNK, and P38 MAP kinase signal pathways. J. Cell. Biochem. 108: 75–86, 2009. © 2009 Wiley‐Liss, Inc.     

TCTP在辐射诱导胶质瘤细胞旁效应中的作用及机制研究

目的:研究肿瘤翻译控制蛋白(TCTP)在辐射诱导胶质瘤细胞旁效应中的作用及机制。方法:给予不同剂量的X射线照射U87、SHG44两种胶质瘤细胞,观察U87以及SHG44细胞的克隆形成率,并在给予最佳照射剂量后,通过Western Blot检测TCTP蛋白表达水平。将经过最佳X射线照射剂量的U87以及SHG44两种胶质瘤细胞与未经过辐射照射的细胞放在一起共培养,通过MTT实验检测胶质瘤细胞的增殖率,Western Blot检测共培养的胶质瘤细胞与经过辐射的胶质瘤细胞中Caspase3蛋白表达水平。结果:U87以及SHG44两种胶质瘤细胞的克隆形成率随着X射线照射剂量增加而显著性降低(P0.05),给予最佳X射线照射剂量后,与未经过X射辐射照射后的细胞相比,其TCTP蛋白表达水平明显升高(P0.05)。经过辐射照射与未经过辐射照射的胶质瘤细胞经过共培养后,与经过辐射的胶质瘤细胞相比,细胞的增殖率明显升高,同时共培养的胶质瘤细胞与经过辐射的胶质瘤细胞相比,Caspase3的蛋白表达明显降低(P0.05)。结论:TCTP的表达增高能够诱导未经过辐射的U87以及SHG44两种胶质瘤细胞的抗凋亡作用增强,其作用机制可能与Caspase3的表达降低有关。     

维生素C联合替莫唑胺对胶质瘤细胞的毒性作用及其机制*

目的:探讨维生素C(VC)联合替莫唑胺(TMZ)对胶质瘤细胞活力的毒性作用及其机制。方法:在体外条件下培养人胶质瘤细胞BMG-1和SHG44细胞,设对照组(不施加VC与TMZ)、TMZ组(0.2 mmol/L)、VC(0.5 mmol/L)+TMZ(0.2 mmol/L)组,TMZ(0.2 mmol/L TMZ)+U0126(10 μmol/L)组,每组实验重复3次。采用MTT实验检测细胞生存率;流式细胞术和Annexin V-FITC/PI染色检测细胞凋亡情况; ROS检测试剂盒检测活性氧簇(ROS)水平, Western blot检测与凋亡、自噬及ERK通路相关蛋白的表达。结果:与对照组比较,TMZ组胶质瘤细胞的存活率显著下降(P＜0.05)。与TMZ组比较,VC+TMZ组胶质细胞瘤细胞的存活率显著下降(P＜0.01),VC+TMZ组中细胞凋亡率显著升高,且Bax、Cleaved caspase-3及Cleaved PARP蛋白表达显著增加,Bcl-2表达显著降低,而ROS水平及细胞自噬率显著降低,LC3-II/LC3-1表达显著降低,p62表达显著增加(P均＜0.05)。同时,联用可降低BMG-1和SHG44细胞中的p-ERK1/2相关蛋白的表达水平,且提高细胞凋亡率(P均＜0.05)。结论:VC联合TMZ能够增强对胶质瘤细胞的毒性,而这一作用是通过ERK信号通路来促进细胞凋亡并抑制替莫唑胺所介导的自噬作用。     

Pro-apoptotic role of integrin β3 in glioma cells

Malignant gliomas are the most destructive type of brain cancer. In order to gain a better understanding of the molecular mechanisms of glioma cell death and survival, we previously established an alkylating agent 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-resistant variant of C6 rat glioma cells. Proteomic analysis indicated a significant down-regulation of integrin beta 3 (ITGB3) in the BCNU-resistant C6R cells. Re-expression of ITGB3 in C6R cells restored the BCNU sensitivity. In U87MG, U373MG, and T98G human glioma cells, there was a positive correlation between ITGB3 expression and the sensitivity to BCNU and etoposide, suggesting an important role of ITGB3 in glioma cell death. Over-expression of ITGB3 cDNA significantly increased the sensitivity of the human glioma cells to the anticancer drug-induced apoptosis. Nitric oxide showed an additive effect on the anticancer drug-induced glioma cell death by increasing ITGB3 expression. Subsequent dissection of signaling pathways indicated that extracellular signal-regulated kinase and unligated integrin-mediated cell death pathway may be involved in the pro-apoptotic role of ITGB3 in glioma cells. These results implicate ITGB3 in glioma cell death/survival and drug resistance.     

Convergent actions of I kappa B kinase beta and protein kinase C delta modulate mRNA stability through phosphorylation of 14-3-3 beta complexed with tristetraprolin

Regulation of gene expression at the level of mRNA stability is a major topic of research; however, knowledge about the regulatory mechanisms affecting the binding and function of AU-rich element (ARE)-binding proteins (AUBPs) in response to extracellular signals is minimal. The beta1,4-galactosyltransferase 1 (beta4GalT1) gene enabled us to study the mechanisms involved in binding of tristetraprolin (TTP) as the stability of its mRNA is regulated solely through one ARE bound by TTP in resting human umbilical vein endothelial cells. Here, we provide evidence that the complex formation of TTP with 14-3-3beta is required to bind beta4GalT1 mRNA and promote its decay. Furthermore, upon tumor necrosis factor alpha stimulation, the activation of both Ikappabeta kinase and protein kinase Cdelta is involved in the phosphorylation of 14-3-3beta on two serine residues, paralleled by release of binding of TTP and 14-3-3beta from beta4GalT1 mRNA, nuclear sequestration of TTP, and beta4GalT1 mRNA stabilization. Thus, a key mechanism regulating mRNA binding and function of the destabilizing AUBP TTP involves the phosphorylation status of 14-3-3beta.     

Apoptosis and involution in the mammary gland are altered in mice lacking a novel receptor, beta1,4-Galactosyltransferase I

Receptor-mediated cell-extracellular matrix (ECM) interactions are critical regulators of cell survival, and perturbing these signaling pathways can disrupt cellular differentiation and function in a variety of tissues, including the mammary gland. One such receptor is the cell surface-associated, long isoform of beta1,4-galactosyltransferase I (GalT I). Deletion of long GalT I leads to increased mammary ductal branching morphogenesis [Dev. Biol., 244 (2002) 114]. Here, we show that this expansion in the mammary epithelial (ME) cell compartment is accomplished through decreased apoptosis during pregnancy and involution. Decreased apoptosis during involution is concomitant with delayed alveolar collapse, persistent expression of the milk protein gene alpha-lactalbumin and delayed expression of genes associated with the tissue-remodeling phase of involution. Using 3-dimensional in vitro cultures, we show that the decrease in apoptosis is dependent on laminin 1, a ligand for surface GalT I, suggesting that surface GalT I negatively influences ECM-dependent cell survival, a novel function for an ECM receptor. In the best-studied examples, ECM promotes survival through integrin receptor-mediated activation of focal adhesion kinase (FAK). Aggregation of surface GalT I also activates FAK, therefore, we asked if FAK activation was altered in ME from long GalT I null mice. Activated FAK was appropriately localized to focal adhesions in long GalT I null ME. However, FAK activation was constitutively reduced 4.5-fold in long GalT I nulls relative to wild type. Expression of the integrin beta1 subunit was not affected by loss of long GalT I. Collectively, these results suggest that surface GalT I might negatively regulate ME cell survival by linking integrin-independent FAK activation to apoptotic rather than survival signaling events.     

LncRNA MYU对胶质瘤细胞周期分布、增殖、转移和凋亡以及PI3K/Akt信号通路的影响

摘要 目的：探讨长链非编码RNA（LncRNA）MYU对胶质瘤细胞周期分布、细胞增殖、迁移、侵袭和凋亡的影响,并初步探讨其作用机制。方法：实时荧光定量PCR（RT-qPCR）检测人脑正常胶质细胞HEB和胶质瘤细胞（U-251MG、A172、SHG139）中LncRNA MYU的表达情况。选取SHG139细胞,分为正常对照（NC）组、si-con组、si-LncRNA MYU组进行转染实验,行RT-qPCR检测转染效果。分别采用流式细胞术、细胞计数试剂盒（CCK-8）、Transwell实验检测沉默LncRNA MYU对SHG139细胞周期分布和凋亡、细胞增殖、细胞迁移和侵袭的影响。蛋白免疫印迹（Western blot）法检测基质金属蛋白酶2（MMP-2）、MMP-9、裂解的半胱氨酸天冬氨酸蛋白酶3（Cleaved caspase-3）、Cleaved caspase-9以及磷脂酰肌醇-3-羟激酶/蛋白激酶B（PI3K/Akt）信号通路相关蛋白表达情况。结果：LncRNA MYU在胶质瘤细胞株中比人脑正常胶质细胞中的表达水平显著升高（P＜0.05）,因此选择表达量最高的SHG139细胞进行转染实验。沉默LncRNA MYU能够显著诱导SHG139细胞G0-G1期阻滞、抑制细胞增殖、迁移和侵袭并诱导细胞凋亡（P＜0.05）。沉默LncRNA MYU可显著抑制MMP-2、MMP-9、p-PI3K和p-AKT表达并促进Cleaved caspase-3、Cleaved caspase-9表达（P＜0.05）。结论：沉默LncRNA MYU可诱导胶质瘤细胞G0-G1期阻滞,抑制细胞增殖、迁移和侵袭,促进细胞凋亡,其机制可能与抑制PI3K/AKT信号通路有关。