Proliferating cell nuclear antigen: a proteomics view

Proliferating cell nuclear antigen (PCNA), a cell cycle marker protein, is well known as a DNA sliding clamp for DNA polymerase delta and as an essential component for eukaryotic chromosomal DNA replication and repair. Due to its mobility inside nuclei, PCNA is dynamically presented in a soluble or chromatin-associated form. The heterogeneity and specific modifications of PCNA may reflect its multiple functions and the presence of many binding partners in the cell. The recent proteomics approaches applied to characterizing PCNA interactions revealed multiple PCNA partners with a wide spectrum of activity and unveiled the possible existence of new PCNA functions. Since more than 100 PCNA-interacting proteins and several PCNA modifications have already been reported, a proteomics point of view seems exactly suitable to better understand the role of PCNA in cellular functions. Received 29 May 2008; received after revision 7 July 2008; accepted 16 July 2008     

无血清分离人结直肠癌细胞的体外培养形态及标志物表达特征

目的探讨人结直肠肿瘤干细胞在体外分化过程中细胞形态和干细胞相关标志物CD133的表达变化,为进一步研究结直肠肿瘤干细胞分化走向提供实验依据。方法取来源于人结直肠癌的细胞系HCT116,无血清培养分离出CD133+细胞,加血清诱导分化,相差显微镜下观察其形态变化;在未分化状态下无血清培养第7天和14天与血清诱导分化后收集细胞,利用流式细胞仪检测干细胞标志物CD133的表达量,采用激光共聚焦检测CD133表面标记分子的表达。结果 1)细胞形态:无血清培养分离的CD133+细胞,在生长过程中聚集成规则的细胞球,血清诱导后即贴壁生长,贴壁形态与同来源细胞形态一致,且再次无血清悬浮培养后聚集成球稳定生长。2)标志物变化:结直肠肿瘤干细胞未分化时CD133表面标记分子高表达,流式细胞仪检测未分化细胞CD133第7天表达率为(20.4±0.52)%,第14天表达率为(78.5±2.80)%,分化后表达率为(0.50±0.17)%。结论细胞形态和标志物表达改变均表明高表达CD133+的HCT116结直肠癌肿瘤干细胞可定向分化为同源的结直肠癌细胞,CD133+细胞经血清诱导后表达下调而使细胞失去干细胞特性。     

Cytomegalovirus infection blocks apoptosis in cancer cells

Recent pathological findings reveal a higher frequency of human cytomegalovirus (HCMV) in tumor cells from different tumors compared with surrounding tissues. Experimental investigations suggest possible supportive effects of HCMV for tumor development and progression. One HCMV effect on tumor cells is the inhibition of apoptosis, leading to the promotion of tumor cell survival. Decreased sensitivity to treatment-induced tumor cell death is a major reason for failure of anticancer chemotherapy. HCMV infection interferes with both the intrinsic and extrinsic cellular apoptosis pathways. HCMV promotes cell survival signaling influencing the tumor suppressor p53 and its relative p73, and stimulates the antiapoptotic Ras/Raf/MEK/Erk- and PI-3K-signaling pathways. Antiapoptotic effects mediated by HCMV are inhibited by antiviral treatment in cell culture. Therefore, a better understanding of the influence of HCMV infection on tumor cell apoptosis might translate into improved anti-cancer therapy.Received 10 November 2003; received after revision 22 December 2003; accepted 14 January 2004     

Acylphosphatase overexpression triggers SH-SY5Y differentiation towards neuronal phenotype

An acylphosphatase (AcPase) overexpression study was carried out on SH-SY5Y neuroblastoma cells, using a green fluorescent fusion protein (AcP-GFP), with GFP acting as a reporter protein. The cellular proliferation rate was significantly reduced by overexpression of AcPase by a factor of ten. In contrast, clones transfected with two inactive AcPase mutants showed a growth rate comparable to control cells. This suggests that AcPase catalyzes the proliferative down-regulation. AcPase-overexpressing clones showed a physiological mortality rate as assessed by an MTT reduction test and by evaluation of necrotic markers. DNA fragmentation analysis and assays of caspase-3 and poly (ADP-ribose) polymerase (PARP)-active fragments showed no evidence of any apoptotic pattern. AcPase overexpression led to a marked increase in PARP activity as well as Bcl-2 content; these are commonly up-regulated during differentiative processes in neuronal cells. In fact, the typical differentiation marker, growth-associated-protein 43, was significantly up-regulated. Microscopic observations also showed a clear increase in the differentiative phenotype in AcPase-overexpressing cells. Our results clearly show that AcPase plays a primary causative role in neuronal differentiation.Received 3 May 2004; accepted 25 May 2004     

Identification of a restriction point at the M/G1 transition in CHO cells

The regulation of cell cycle progression in normal mammalian cells is dependent on the presence of growth factors. In their absence, non-transformed cells will stop dividing and enter the quiescent state (G0). We show here that in Chinese hamster ovary cells, at least two serum-dependent points exist during G1 that lead to different cellular responses. The first point is located immediately after mitosis and is suggested to link with apoptosis. The second point is located late in G1, and probably corresponds with the classic restriction point R. Cells depleted of serum after the first restriction point will not stop randomly in G1 but continue G1 progression until they reach the late restriction point, as marked by translocation of p42^MAPkinase (ERK2) to the nucleus.Received 18 September 2003; received after revision 11 December 2003; accepted 19 December 2003     

Medium- and short-chain dehydrogenase/reductase gene and protein families

Alcohol dehydrogenase 3 (ADH3) is highly conserved, ubiquitously expressed in mammals and involved in essential cellular pathways. A large active site pocket entails special substrate specificities: shortchain alcohols are poor substrates, while medium-chain alcohols and particularly the glutathione adducts S-hydroxymethylglutathione (HMGSH) and S-nitrosoglutathione (GSNO) are efficiently converted under concomitant use of NAD⁺/NADH. By oxidation of HMGSH, the spontaneous glutathione adduct of formaldehyde, ADH3 is implicated in the detoxification of formaldehyde. Through the GSNO reductase activity, ADH3 can affect the transnitrosation equilibrium between GSNO and S-nitrosated proteins, arguing for an important role in NO homeostasis. Recent findings suggest that ADH3-mediated GSNO reduction and subsequent product formation responds to redox states in terms of NADH availability and glutathione levels. Finally, a dual function of ADH3 is discussed in view of its potential implications for asthma.     

Cofilin activity during insulin-like growth factor I-stimulated neuroblastoma cell motility

Insulin-like growth factor I (IGF-I) is a potent stimulator of neuroblastoma cell motility. Cell motility requires lamellipodium extension at the leading edge of the cell through organized actin polymerization, and IGF-I stimulates lamellipodial elaboration in human neuroblastoma cells. Rac is a Rho GTPase that stimulates lamellipodial formation via the regulation of actin polymerization. In this study, we show that IGF-I-stimulated phosphatidylinositol 3-kinase (PI-3K) activity promotes rac activation and subsequent activation of the down- stream effectors LIM kinase and cofilin. Overexpression of wild-type LIM kinase and wild-type Xenopus ADF/cofilin (XAC) suppresses IGF-I-stimulated motility in SH-SY5Y cells, while expression of dominant negative LIM kinase and constitutively active XAC increases SH-SY5Y motility in the absence of IGF-I stimulation. These results suggest that regulation by cofilin of actin depolymerization is important in the process of neuroblastoma cell motility, and IGF-I regulates cofilin activity in part through PI-3K, rac, and LIM kinase.Received 18 October 2004; received after revision 3 December 2004; accepted 16 December 2004     

Mast cell tryptase,a still enigmatic enzyme

Tryptases constitute a subfamily of trypsin-like proteinases, stored in the mast cell secretory granules of all mammalian organisms. These enzymes are released along with other mediators into the extracellular medium upon mast cell activation/degranulation. Among the trypsin-like enzymes, tryptases are unique: they are present as active enzymes in the mast cell granules, but display activity only extracellularly, and have a specificity which is much more restricted than trypsin. Tryptases are mostly tetrameric, and in only few organisms (not in humans) are they inhibited by endogenous inhibitors in vitro. The enzymatic and molecular properties of tryptases are far better characterized that any of their plausible biological functions. On the basis of its structural and functional features it could be predicted that tryptase would not degrade a large number of proteins in vivo due to low accessibility to the tetramer central pore where the active sites face inwards. Although their biological function has not yet been clarified, tryptases seem to be involved in a number of mast cell-mediated allergic and inflammatory diseases. In particular, the involvement of tryptase in asthma, an inflammatory disease of the airways often caused by allergy, has been proposed. Here we review the present knowledge on the structure-function relationship of tryptases from different organisms, with special emphasis on human enzymes, and on their role in a variety of pathophsyiological processes.Received 29 October 2003; received after revision 3 December 2003; accepted 11 December 2003     

RNA cytosine methyltransferase Nsun3 regulates embryonic stem cell differentiation by promoting mitochondrial activity

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNA^Met. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNA^Met methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.     

Cell surface antigen profiling using a novel type of antibody array immobilised to plasma ion-implanted polycarbonate

To identify and sort out subpopulations of cells from more complex and heterogeneous assemblies of cells is important for many biomedical applications, and the development of cost- and labour-efficient techniques to accomplish this is warranted. In this report, we have developed a novel array-based platform to discriminate cellular populations based on differences in cell surface antigen expressions. These cell capture microarrays were produced through covalent immobilisation of CD antibodies to plasma ion immersion implantation-treated polycarbonate (PIII-PC), which offers the advantage of a transparent matrix, allowing direct light microscopy visualisation of captured cells. The functionality of the PIII-PC array was validated using several cell types, resulting in unique surface antigen expression profiles. PIII-PC results were compatible with flow cytometry, nitrocellulose cell capture arrays and immunofluorescent staining, indicating that the technique is robust. We report on the use of this PIII-PC cluster of differentiation (CD) antibody array to gain new insights into neural differentiation of mouse embryonic stem (ES) cells and into the consequences of genetic targeting of the Notch signalling pathway, a key signalling mechanism for most cellular differentiation processes. Specifically, we identify CD98 as a novel marker for neural precursors and polarised expression of CD9 in the apical domain of ES cell-derived neural rosettes. We further identify expression of CD9 in hitherto uncharacterised non-neural cells and enrichment of CD49e- and CD117-positive cells in Notch signalling-deficient ES cell differentiations. In conclusion, this work demonstrates that covalent immobilisation of antibody arrays to the PIII-PC surface provides faithful cell surface antigen data in a cost- and labour-efficient manner. This may be used to facilitate high throughput identification and standardisation of more precise marker profiles during stem cell differentiation and in various genetic and disease contexts.     

Inactivation of Ret/Ptc1 oncoprotein and inhibition of papillary thyroid carcinoma cell proliferation by indolinone RPI-1

Genetic alterations causing oncogenic activation of the RET gene are recognized as pathogenic events in papillary and medullary thyroid carcinomas. Inhibition of Ret oncoprotein functions could thereby represent a specific therapeutic approach. We previously described the inhibitory activity of the 2-indolinone derivative RPI-1 (formerly Cpd1) on the tyrosine kinase activity and transforming ability of the products of the RET/PTC1 oncogene exogenously expressed in murine cells. In the present study, we investigated the effects of RPI-1 in the human papillary thyroid carcinoma cell line TPC-1 spontaneously harboring the RET/PTC1 rearrangement. Treatment with RPI-1 inhibited cell proliferation and induced accumulation of cells at the G2 cell cycle phase. In treated cells, Ret/Ptc1 tyrosine phosphorylation was abolished along with its binding to Shc and phospholipase C, thereby indicating abrogation of constitutive signaling mediated by the oncoprotein. Activation of JNK2 and AKT was abolished, thus supporting the drug inhibitory efficacy on downstream pathways. In addition, cell growth inhibition was associated with a reduction in telomerase activity by nearly 85%. These findings in a cellular context relevant to the pathological function of RET oncogenes support the role of Ret oncoproteins as useful targets for therapeutic intervention, and suggest RPI-1 as a promising candidate for preclinical development in the treatment of thyroid tumors expressing RET oncogenes.Received 31 December 2002; received after revision 21 February 2003; accepted 10 April 2003     

Molecular and cellular mechanisms of T Cell development

The thymus is central to the establishment of a functioning immune system. Here is the place where T cells mature from hematopoietic progenitors, driven by mutual interactions of stromal cells and the developing thymocytes. As a result, different types of T cells are generated, all of which have been carefully selected for the ability to act in host defense towards non-self and against the potential to mount pathogenic self-reactive autoimmune responses. In this review we summarize our present knowlege on the lineage decisions taking place during this development, the selection processes responsible for shaping the T cell antigen-receptor repertoire, the interactions with the stromal components and the signal transduction pathways which transform the interactions with the thymic microenvironment into cellular responses of survival, proliferation, differentiation and, importantly, also of cell death. Received 12 June 2003; received after revision 22 July 2003; accepted 28 July 2003     

cAMP initiates early phase neuron-like morphology changes and late phase neural differentiation in mesenchymal stem cells

The intracellular second messenger cAMP is frequently used in induction media to induce mesenchymal stem cells (MSCs) into neural lineage cells. To date, an understanding of the role cAMP exerts on MSCs and whether cAMP can induce MSCs into functional neurons is still lacking. We found cAMP initiated neuron-like morphology changes early and neural differentiation much later. The early phase changes in morphology were due to cell shrinkage, which subsequently rendered some cells apoptotic. While the morphology changes occurred prior to the expression of neural markers, it is not required for neural marker expression and the two processes are differentially regulated downstream of cAMP-activated protein kinase A. cAMP enabled MSCs to gain neural marker expressions with neuronal function, such as, calcium rise in response to neuronal activators, dopamine, glutamate, and potassium chloride. However, only some of the cells induced by cAMP responded to the three neuronal activators and further lack the neuronal morphology, suggesting that although cAMP is able to direct MSCs towards neural differentiation, they do not achieve terminal differentiation.     

Cbl signaling networks in the regulation of cell function

Cbl proteins control multiple cellular processes by acting as ubiquitin ligases and multifunctional adaptor molecules. They are involved in the control of cell proliferation, differentiation and cell morphology, as well as in pathologies such as autoimmune diseases, inflammation and cancer. Here we review recent advances in understanding the role of Cbl and the importance of a growing repertoire of Cbl-interacting proteins in the regulation of signaling pathways triggered by growth factors, antigens, cell adhesion, cytokines and hormones. We also address key issues of the nature of proteins that bind Cbl in particular cells, where they are located, and how they are altered or traffic within cells upon stimulation. It is becoming obvious that temporal and spatial changes in Cbl signaling networks are essential for the control of physiological processes in a variety of cells and organs and that their deregulation can result in the development of human diseases.Received 22 January 2003; received after revision 11 March 2003; accepted 26 March 2003