Epstein-Barr Virus Latent Membrane Protein 2 Effects on Epithelial Acinus Development Reveal Distinct Requirements for the PY and YEEA motifs

Epstein-Barr virus (EBV) is a gammaherpesvirus associated with numerous cancers, including the epithelial cancers nasopharyngeal carcinoma (NPC) and gastric carcinoma. The latent membrane protein 2 (LMP2) encoded by EBV is consistently detected in NPC tumors and promotes a malignant phenotype when expressed in epithelial cells by inducing transformation and migration and inhibiting differentiation. Grown in three dimensions (3D) on Matrigel, the nontumorigenic mammary epithelial cell line MCF10A forms hollow, spherical acinar structures that maintain normal glandular features. Expression of oncogenes in these cells allows for the study of multiple aspects of tumor development in a 3D culture system. This study sought to examine the effects of LMP2 on the generation of MCF10A acini. LMP2 expression induced abnormal acini that were large, misshapen, and filled, indicating that LMP2 induced proliferation, impaired cellular polarization, and induced resistance to cell death, leading to luminal filling. Induction of cell death resistance required the PY, immunoreceptor tyrosine activation motif (ITAM), and YEEA signaling domains of LMP2 and activation of the Src and Akt signaling pathways. The PY domain was required for the inhibition of anoikis and also the delayed proliferative arrest of the LMP2-expressing cells. In addition to directly altering acinus formation, expression of LMP2 also induced morphological and protein expression changes consistent with epithelial-mesenchymal transition (EMT) in a manner that required only the YEEA signaling motif of LMP2. These findings indicate that LMP2 has considerable transforming properties that are not evident in standard tissue culture and requires the ability of LMP2A to bind ubiquitin ligases and Src family kinases.     

Epstein-Barr Virus Latent Membrane Protein 2A Contributes to Anoikis Resistance through ERK Activation

Epstein-Barr virus (EBV) is associated with various malignancies, including epithelial cancers. In this study, we analyzed the effect of EBV infection on epithelial cells by using EBV-converted epithelial cells. In EBV-positive cells, the extracellular signal-regulated kinase (ERK) pathway is constitutively activated. Inhibition of ERK activity leads to reduced anoikis resistance; therefore, EBV-positive cells are more resistant to anoikis, a type of apoptosis induced by cell detachment, than are EBV-negative cells. Among the viral genes expressed in EBV-positive cells, the latent membrane protein 2A (LMP2A) is responsible for induction of ERK-mediated anoikis resistance, although the expression level of LMP2A is much lower in EBV-positive cells than in EBV-transformed B cells. Further analysis demonstrated that LMP2A downregulation of the proanoikis mediator Bim through proteasomal degradation is dependent on the immunoreceptor tyrosine-based activation motif (ITAM). These findings suggest that LMP2A-mediated ERK activation is involved in the generation of EBV-associated epithelial malignancies.     

Detachment-induced autophagy during anoikis and lumen formation in epithelial acini

The individual units (called acini) comprising glandular epithelium possess a hollow lumen that is filled in early epithelial cancers. While investigating the generation of this hollow lumen using an in vitro three-dimensional (3D) epithelial cell culture model, we observed extensive autophagy in dying cells during lumen formation, and thus, proposed that excessive self-eating may promote cell death in the lumen. However, we now reassess this hypothesis. Because cells in the lumen die due to extracellular matrix (ECM) deprivation (anoikis), we tested if autophagy is regulated by ECM detachment. We discovered that detachment strongly induces autophagy in epithelial cells, even when apoptosis is inhibited by Bcl-2 expression. RNAi-mediated depletion of autophagy-related (ATG) genes inhibits detachment-induced autophagy, resulting in increased apoptosis and reduced clonogenic recovery following anoikis. Similarly, during 3D morphogenesis, ATG-depletion enhances luminal apoptosis and fails to elicit long-term luminal survival and filling, even when combined with apoptotic inhibition. Thus, autophagy promotes epithelial cell survival during anoikis. These results broach the possibility that autophagy contributes to the survival of tumor cells lacking appropriate matrix contact, either during early carcinoma formation or in the later stages of dissemination and metastasis.     

Induction of autophagy during extracellular matrix detachment promotes cell survival

Autophagy has been proposed to promote cell death during lumen formation in three-dimensional mammary epithelial acini because numerous autophagic vacuoles are observed in the dying central cells during morphogenesis. Because these central cells die due to extracellular matrix (ECM) deprivation (anoikis), we have directly interrogated how matrix detachment regulates autophagy. Detachment induces autophagy in both nontumorigenic epithelial lines and in primary epithelial cells. RNA interference-mediated depletion of autophagy regulators (ATGs) inhibits detachment-induced autophagy, enhances apoptosis, and reduces clonogenic recovery after anoikis. Remarkably, matrix-detached cells still exhibit autophagy when apoptosis is blocked by Bcl-2 overexpression, and ATG depletion reduces the clonogenic survival of Bcl-2-expressing cells after detachment. Finally, stable reduction of ATG5 or ATG7 in MCF-10A acini enhances luminal apoptosis during morphogenesis and fails to elicit long-term luminal filling, even when combined with apoptotic inhibition mediated by Bcl-2 overexpression. Thus, autophagy promotes epithelial cell survival during anoikis, including detached cells harboring antiapoptotic lesions.     

DNA methylation control of tissue polarity and cellular differentiation in the mammary epithelium

Alterations in gene expression accompany cell-type-specific differentiation. In complex systems where functional differentiation depends on the organization of specific cell types into highly specialized structures (tissue morphogenesis), it is not known how epigenetic mechanisms that control gene expression influence this stepwise differentiation process. We have investigated the effect of DNA methylation, a major epigenetic pathway of gene silencing, on the regulation of mammary acinar differentiation. Our in vitro model of differentiation encompasses human mammary epithelial cells that form polarized and hollow tissue structures (acini) when cultured in the presence of basement membrane components. We found that acinar morphogenesis was accompanied with chromatin remodeling, as shown by alterations in histone 4 acetylation, heterochromatin 1 protein, and histone 3 methylated on lysine 9, and with an increase in expression of MeCP2, a mediator of DNA-methylation-induced gene silencing. DNA hypomethylation induced by treatment with 5-aza-2' deoxycytidine during acinar differentiation essentially prevented the formation of apical tissue polarity. This treatment also induced the expression of CK19, a marker of cells that are in a transitional differentiation stage. These results suggest that DNA methylation is a mechanism by which mammary epithelial differentiation is coordinated both at the tissue and cellular levels.     

Epstein-Barr Virus Latent Membrane Protein-2A Induces ITAM/Syk- and Akt-Dependent Epithelial Migration through αV-Integrin Membrane Translocation

Epstein-Barr virus (EBV) is a highly prevalent herpesvirus associated with epithelial cancers, including nasopharyngeal carcinoma (NPC). The EBV protein latent membrane protein 2 (LMP2) is expressed in NPC tumor tissue and has been shown to induce transformation, inhibit differentiation, and promote migration of epithelial cells. In this study, the effect of LMP2A on migration of human epithelial cells was further analyzed. LMP2A expression induced migration in human foreskin keratinocytes (HFK) and HaCaT keratinocytes measured by wound healing scratch assay and chemoattractant-induced Transwell migration assay. The induction of migration by LMP2A required the ITAM signaling domain of LMP2A and activation of the Syk tyrosine kinase. LMP2A-induced Transwell migration required the Akt signaling pathway, and activation of Akt by LMP2A required the ITAM signaling domain of LMP2A. LMP2A also induced phosphorylation of the Akt target GSK3β, a Wnt signaling mediator that has been shown to regulate the activity of focal adhesion kinase (FAK), a tyrosine kinase activated by clustering and ligand interaction of integrins. Inhibition of either FAK or its signaling mediator Src kinase inhibited LMP2A-induced migration. Interestingly, αV-integrin was greatly increased in membrane-enriched fractions by LMP2A, and a neutralizing antibody to αV-integrin blocked migration, suggesting that the effects of LMP2A on membrane-localized αV-integrin promoted migration. The results of this study indicate that LMP2A expression in human epithelial cells induces αV-integrin-dependent migration through a mechanism requiring ITAM-mediated Syk and Akt activation and inducing membrane translocation or stabilization of αV-integrin and FAK activation. The specific effects of LMP2A on an integrin with a diverse repertoire of ligand specificities could promote migration of different cell types and be initiated by multiple chemoattractants.     

A Computational Study of the Development of Epithelial Acini: I. Sufficient Conditions for the Formation of a Hollow Structure

Normal hollow epithelial acini are 3-dimensional culture structures that resemble the architecture and functions of normal breast glands and lobules. This experimental model enables in vitro investigations of genotypic and molecular abnormalities associated with epithelial cancers. However, the way in which the acinar structure is formed is not yet completely understood. Gaining more information about consecutive stages of acini development—starting from a single cell that gives rise to a cluster of randomly oriented cells, followed by cell differentiation that leads to a layer of polarised cells enclosing the hollow lumen—will provide insight into the transformations of eukaryotic cells that are necessary for their successful arrangement into an epithelium. In this paper, we introduce a two-dimensional single-cell-based model representing the cross section of a typical acinus. Using this model, we investigate mechanisms that lead to the unpolarised cell growth, cell polarisation, stabilisation of the acinar structure and maintenance of the hollow lumen and discuss the sufficient conditions for each stage of acinar formation. In the follow-up paper (Rejniak and Anderson, A computational study of the development of epithelial acini. II. Necessary conditions for structure and lumen stability), we investigate what morphological changes are observable in the growing acini when some assumptions of this model are relaxed.     

Diindolylmethane-mediated Gli1 Protein Suppression Induces Anoikis in Ovarian Cancer Cells in Vitro and Blocks Tumor Formation Ability in Vivo

Anoikis is a cell death that occurs due to detachment of a cell from the extracellular matrix (ECM). Resistance to anoikis is a primary feature of a cell that undergoes metastasis. In this study for the first time, we demonstrated the potential role of Gli1 in anoikis resistance. Treatment of various ovarian cancer cells by different concentrations of diindolylmethane (DIM), an active ingredient of cruciferous vegetables, reduced the anoikis resistance in a concentration-dependent manner. Reduction in anoikis resistance was associated with a decrease in the expression of Gli1 and an increase in the cleavage of poly(ADP-ribose) polymerase (PARP). Sonic hedgehog (Shh) treatment not only increased the expression of Gli1, but also blocked anoikis induced by DIM and abrogated the change in the expression of Gli1 and cleaved PARP by DIM. To confirm the role of Gli1, hedgehog inhibitor cyclopamine, Gli1 siRNA and Gli1(-/-) mouse embryonic fibroblasts (MEFs) were used. Cyclopamine treatment alone significantly reduced anoikis resistance in A2780 and OVCAR-429 cells. Cyclopamine-mediated reduction in anoikis resistance was associated with reduced expression of Gli1 and induction of cleaved PARP. Shh treatment blocked cyclopamine-induced anoikis. Silencing Gli1 expression induced anoikis and cleavage of PARP in A2780 and OVCAR-429 cells. Furthermore, Gli1(-/-) MEFs were more sensitive to anoikis compared with Gli1(+/+) MEFs. Our in vivo studies established that DIM- or cyclopamine-treated ovarian cancer cells under suspension culture conditions drastically lost their ability of tumor formation in vivo in mice. Taken together, our results establish that Gli1 is a critical player in anoikis resistance in ovarian cancer.     

Autophagy Activation Is Involved in 3,4-Methylenedioxymethamphetamine (‘Ecstasy’)—Induced Neurotoxicity in Cultured Cortical Neurons

Autophagic (type II) cell death, characterized by the massive accumulation of autophagic vacuoles in the cytoplasm of cells, has been suggested to play pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is an illicit drug causing long-term neurotoxicity in the brain. Apoptotic (type I) and necrotic (type III) cell death have been implicated in MDMA-induced neurotoxicity, while the role of autophagy in MDMA-elicited neurotoxicity has not been investigated. The present study aimed to evaluate the occurrence and contribution of autophagy to neurotoxicity in cultured rat cortical neurons challenged with MDMA. Autophagy activation was monitored by expression of microtubule-associated protein 1 light chain 3 (LC3; an autophagic marker) using immunofluorescence and western blot analysis. Here, we demonstrate that MDMA exposure induced monodansylcadaverine (MDC)- and LC3B-densely stained autophagosome formation and increased conversion of LC3B-I to LC3B-II, coinciding with the neurodegenerative phase of MDMA challenge. Autophagy inhibitor 3-methyladenine (3-MA) pretreatment significantly attenuated MDMA-induced autophagosome accumulation, LC3B-II expression, and ameliorated MDMA-triggered neurite damage and neuronal death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in neurons and aggravated neurite degeneration, indicating that excessive autophagosome accumulation contributes to MDMA-induced neurotoxicity. Furthermore, MDMA induced phosphorylation of AMP-activated protein kinase (AMPK) and its downstream unc-51-like kinase 1 (ULK1), suggesting the AMPK/ULK1 signaling pathway might be involved in MDMA-induced autophagy activation.     

Hugl1 and Hugl2 in Mammary Epithelial Cells: Polarity,Proliferation, and Differentiation

Loss of epithelial polarity is described as a hallmark of epithelial cancer. To determine the role of Hugl1 and Hugl2 expression in the breast, we investigated their localization in human mammary duct tissue and the effects of expression modulation in normal and cancer cell lines on polarity, proliferation and differentiation. Expression of Hugl1 and Hugl2 was silenced in both MCF10A cells and Human Mammary Epithelial Cells and cell lines were grown in 2-D on plastic and in 3-D in Matrigel to form acini. Cells in monolayer were compared for proliferative and phenotypic changes while acini were examined for differences in size, ability to form a hollow lumen, nuclear size and shape, and localization of key domain-specific proteins as a measure of polarity. We detected overlapping but distinct localization of Hugl1 and Hugl2 in the human mammary gland, with Hugl1 expressed in both luminal and myoepithelium and Hugl2 largely restricted to myoepithelium. On a plastic surface, loss of Hugl1 or Hugl2 in normal epithelium induced a mesenchymal phenotype, and these cells formed large cellular masses when grown in Matrigel. In addition, loss of Hugl1 or Hugl2 expression in MCF10A cells resulted in increased proliferation on Matrigel, while gain of Hugl1 expression in tumor cells suppressed proliferation. Loss of polarity was also observed with knockdown of either Hugl1 or Hugl2, with cells growing in Matrigel appearing as a multilayered epithelium, with randomly oriented Golgi and multiple enlarged nuclei. Furthermore, Hugl1 knock down resulted in a loss of membrane identity and the development of cellular asymmetries in Human Mammary Epithelial Cells. Overall, these data demonstrate an essential role for both Hugl1 and Hugl2 in the maintenance of breast epithelial polarity and differentiated cell morphology, as well as growth control.     

Restoring diabetes-induced autophagic flux arrest in ischemic/reperfused heart by ADIPOR (adiponectin receptor) activation involves both AMPK-dependent and AMPK-independent signaling

Macroautophagy/autophagy is increasingly recognized as an important regulator of myocardial ischemia-reperfusion (MI-R) injury. However, whether and how diabetes may alter autophagy in response to MI-R remains unknown. Deficiency of ADIPOQ, a cardioprotective molecule, markedly increases MI-R injury. However, the role of diabetic hypoadiponectinemia in cardiac autophagy alteration after MI-R is unclear. Utilizing normal control (NC), high-fat-diet-induced diabetes, and Adipoq knockout (adipoq^?/?) mice, we demonstrated that autophagosome formation was modestly inhibited and autophagosome clearance was markedly impaired in the diabetic heart subjected to MI-R. adipoq^?/? largely reproduced the phenotypic alterations observed in the ischemic-reperfused diabetic heart. Treatment of diabetic and adipoq^?/? mice with AdipoRon, a novel ADIPOR (adiponectin receptor) agonist, stimulated autophagosome formation, markedly increased autophagosome clearance, reduced infarct size, and improved cardiac function (P < 0.01 vs vehicle). Mechanistically, AdipoRon caused significant phosphorylation of AMPK-BECN1 (Ser93/Thr119)-class III PtdIns3K (Ser164) and enhanced lysosome protein LAMP2 expression both in vivo and in isolated adult cardiomyocytes. Pharmacological AMPK inhibition or genetic Prkaa2 mutation abolished AdipoRon-induced BECN1 (Ser93/Thr119)-PtdIns3K (Ser164) phosphorylation and AdipoRon-stimulated autophagosome formation. However, AdipoRon-induced LAMP2 expression, AdipoRon-stimulated autophagosome clearance, and AdipoRon-suppressed superoxide generation were not affected by AMPK inhibition. Treatment with MnTMPyP (a superoxide scavenger) increased LAMP2 expression and stimulated autophagosome clearance in simulated ischemic-reperfused cardiomyocytes. However, no additive effect between AdipoRon and MnTMPyP was observed. Collectively, these results demonstrate that hypoadiponectinemia impairs autophagic flux, contributing to enhanced MI-R injury in the diabetic state. ADIPOR activation restores AMPK-mediated autophagosome formation and antioxidant-mediated autophagosome clearance, representing a novel intervention effective against MI-R injury in diabetic conditions.     

Role of the TRAF Binding Site and NF-κB Activation in Epstein-Barr Virus Latent Membrane Protein 1-Induced Cell Gene Expression

In this study, we investigated the induction of cellular gene expression by the Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1). Previously, LMP1 was shown to induce the expression of ICAM-1, LFA-3, CD40, and EBI3 in EBV-negative Burkitt lymphoma (BL) cells and of the epidermal growth factor receptor (EGF-R) in epithelial cells. We now show that LMP1 expression also increased Fas and tumor necrosis factor receptor-associated factor 1 (TRAF1) in BL cells. LMP1 mediates NF-κB activation via two independent domains located in its C-terminal cytoplasmic tail, a TRAF-interacting site that associates with TRAF1, -2, -3, and -5 through a PXQXT/S core motif and a TRADD-interacting site. In EBV-transformed B cells or transiently transfected BL cells, significant amounts of TRAF1, -2, -3, and -5 are associated with LMP1. In epithelial cells, very little TRAF1 is expressed, and only TRAF2, -3, and -5, are significantly complexed with LMP1. The importance of TRAF binding to the PXQXT/S motif in LMP1-mediated gene induction was studied by using an LMP1 mutant that contains alanine point mutations in this motif and fails to associate with TRAFs. This mutant, LMP1(P204A/Q206A), induced 60% of wild-type LMP1 NF-κB activation and had approximately 60% of wild-type LMP1 effect on Fas, ICAM-1, CD40, and LFA-3 induction. In contrast, LMP1(P204A/Q206A) was substantially more impaired in TRAF1, EBI3, and EGF-R induction. Thus, TRAF binding to the PXQXT/S motif has a nonessential role in up-regulating Fas, ICAM-1, CD40, and LFA-3 expression and a critical role in up-regulating TRAF1, EBI3, and EGF-R expression. Further, D1 LMP1, an LMP1 mutant that does not aggregate failed to induce TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 expression confirming the essential role for aggregation in LMP1 signaling. Overexpression of a dominant form of IκBα blocked LMP1-mediated TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 up-regulation, indicating that NF-κB is an important component of LMP1-mediated gene induction from both the TRAF- and TRADD-interacting sites.     

Suppression of Autophagy Enhanced Growth Inhibition and Apoptosis of Interferon-β in Human Glioma Cells

Interferon-beta (IFN-β) is a cytokine with anti-viral, anti-proliferative, and immunomodulatory effects. In this study, we investigated the effects of IFN-β on the induction of autophagy and the relationships among autophagy, growth inhibition, and apoptosis induced by IFN-β in human glioma cells. We found that IFN-β induced autophagosome formation and conversion of microtubule associated protein 1 light chain 3 (LC3) protein, whereas it inhibited cell growth through caspase-dependent cell apoptosis. The Akt/mTOR signaling pathway was involved in autophagy induced by IFN-β. A dose- and time-dependent increase of p-ERK 1/2 expression was also observed in human glioma cells treated with IFN-β. Autophagy induced by IFN-β was suppressed when p-ERK1/2 was impaired by treatment with U0126. We also demonstrated that suppression of autophagy significantly enhanced growth inhibition and cell apoptosis induced by IFN-β, whereas inhibition of caspase-dependent cell apoptosis impaired autophagy induced by IFN-β. Collectively, these findings indicated that autophagy induced by IFN-β was associated with the Akt/mTOR and ERK 1/2 signaling pathways, and inhibition of autophagy could enhance the growth inhibitory effects of IFN-β and increase apoptosis in human glioma cells. Together, these findings support the possibility that autophagy inhibitors may improve IFN-β therapy for gliomas.     

Latent membrane protein 2A inhibits transforming growth factor-beta 1-induced apoptosis through the phosphatidylinositol 3-kinase/Akt pathway

Latent membrane protein 2A (LMP2A) blocks B-cell receptor signal transduction in vitro by binding the Syk and Lyn protein tyrosine kinases. As well as blocking B-cell signal transduction, LMP2A has been shown to activate the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, which acts as a survival signal in both B cells and epithelial cells. Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine that plays important roles in regulating cell growth and differentiation in many biological systems. The loss of the growth-inhibitory response to the TGF-beta 1 signal is found in many cancers and is widely thought to promote tumor development. In this study, we found that LMP2A induced the phosphorylation of Akt (serine 473) in Burkitt's lymphoma cell line Ramos and in gastric carcinoma cell line HSC-39 and partially enhanced cell viability following TGF-beta 1 treatment. In addition, LMP2A partially inhibited TGF-beta 1-induced DNA fragmentation and cleavage of poly(ADP-ribose) polymerase (PARP). In the presence of LY294002, an inhibitor of PI3-K, the LMP2A-mediated inhibitory effects on TGF-beta 1-induced DNA fragmentation and cleavage of PARP were alleviated. Furthermore, LMP2A did not alter the levels of expression of type I and type II TGF-beta 1 receptors. Taken together, these results suggest that LMP2A may inhibit TGF-beta 1-mediated apoptosis through activation of the PI3-K/Akt pathway.     

鼻咽癌细胞中EB病毒编码的潜伏膜蛋白1活化cyclinD1的表达

为了探讨EB病毒编码的潜伏膜蛋白1(EBV-LMP1)促进细胞增殖,参与EBV相关疾病致瘤的分子机制,研究了LMP1在鼻咽癌细胞中调节cyclinD1表达,进而影响细胞周期行进及细胞恶性表型改变,并初步确定了LMP1发挥该功能的结构域.利用已建株的Tet-on-LMP1-HNE2鼻咽癌细胞系,蛋白质印迹实验分析LMP1诱导cyclinD1蛋白质表达的表达动力学,包括时间效应及剂量效应;利用三种LMP1功能区缺失的突变体及野生型LMP1,以载体型细胞为对照,确定LMP1活化cyclinD1表达的结构域.同时结合基因诱导表达及反义寡聚核酸技术阻断基因表达的实验方法,进一步确定LMP1上调的cyclinD1功能,即对细胞周期行进及细胞恶性表型的影响.结果表明LMP1确实可以诱导cyclinD1的表达（2～4倍）,且诱导具有时间依赖性及剂量依赖性;利用三种LMP1功能区缺失的突变体及野生型LMP1,以载体型细胞为对照,结合报道基因分析法,确定与空白载体细胞系比较,野生型LMP1从转录水平可反式激活cyclinD1报道基因活性约11.2倍,其中CTAR1及CTAR2均可活化cyclinD1表达,但以CTAR2为主,与野生型LMP1诱导cyclinD1反式激活活性比较,CTAR1缺失导致cyclinD1报道基因活性下降23.6%,CTAR2缺失导致cyclinD1活性下降约80.7%,C端均缺失时cyclinD1活性只有野生型的17.7%.流式细胞仪分析显示,强力霉素诱导后cyclinD1高表达的细胞停留于G0/G1期明显减少,较未经诱导的细胞,从66.42%减至56.55%,而进入S期及G2/M期的细胞明显增多.在稳定表达LMP1的细胞中,与导入正义LMP1比较,导入反义LMP1 PS-ODNs及反义cylinD1,可以使细胞软琼脂集落形成率明显降低(从30.48%分别降至15.21%,21.76%).EBV-LMP1可以活化cyclinD1的表达,且发挥这种功能的结构域以CTAR2为主,活化的cyclinD1参与细胞周期行进,抑制LMP1及cyclinD1的表达均可导致细胞软琼脂集落形成率降低.     

Changes in proteasome expression and activity during differentiation of neuronal precursor NTera 2 clone D1 cells

The effect of differentiation of the human neuronal progenitor cell line NTera 2 clone D1 (NT2/D1) by retinoic acid on components of the proteasome system was studied. The chymotrypsin-like and peptidylglutamyl peptide bond hydrolyzing activities of the proteasome increased five weeks after retinoic acid, and following treatment with mitotic inhibitors returned to levels detected in non-differentiated cells. A selective induction of the MHC class II region encoded LMP7 and LMP2 proteasome subunits occurred during differentiation, whereas there were no changes in the expression of the constitutive LMP2 counterpart (delta-subunit) or the constitutive C2 subunit. Immunofluorescence revealed marked LMP7 accumulation in fully differentiated cells, with no changes in the labeling pattern of the constitutive proteasome antigens. The expression of the alpha-subunit of the PA28 proteasome activator was down-regulated in fully differentiated neurons, but was not correlated with changes in enzymatic activity. Changes in proteasome activity and composition may contribute to the processes leading to differentiation of human neurons in vitro and to the properties of fully differentiated neurons.