The budding yeast Pichia pastoris has a novel Sec23p homolog

In Pichia pastoris, coat protein complex II (COPII) vesicles form at discrete transitional ER (tER) sites. Analyzing COPII coat proteins in this yeast will help to reveal the mechanisms of tER organization. Here, we show that like Saccharomyces cerevisiae, P. pastoris contains essential SEC23 and SEC24 genes, as well as the non-essential SEC24 homolog LST1. In addition, P. pastoris contains a novel non-essential SEC23 homolog that we have designated SHL23. The products of all four genes are concentrated at tER sites. Deletion of SHL23 does not disrupt tER morphology. As judged by two-hybrid analysis, Sec23p associates with both Sec24p and Lst1p, whereas Shl23p associates selectively with Lst1p. These results suggest that P. pastoris COPII vesicles contain an Shl23p/Lst1p complex that is absent in S. cerevisiae.     

cDNA macroarray analysis of gene expression in ineffective nodules induced on the Lotus japonicus sen1 mutant

The Lotus japonicus sen1 mutant forms ineffective nodules in which development is arrested at the stage of bacterial differentiation into nitrogen-fixing bacteroids. Here, we used cDNA macroarray systems to compare gene expression in ineffective nodules induced on the sen1 mutant with gene expression in wild-type nodules, in order to identify the host plant genes that are involved in nitrogen fixation. Macroarray analysis coupled with Northern blot analysis revealed that the expression of 18 genes was significantly enhanced in ineffective sen1 nodules, whereas the expression of 30 genes was repressed. Many of the enhanced genes encoded hydrolase enzymes, such as cysteine proteinase and asparaginase, that might function in the early senescence of sen1 nodules. By contrast, the repressed genes encoded nodulins, enzymes that are involved in carbon and nitrogen metabolism, membrane transporters, enzymes involved in phytohormone metabolism and secondary metabolism, and regulatory proteins. These proteins might have a role in the establishment of nitrogen fixation. In addition, we discovered two novel genes that encoded glutamate-rich proteins and were localized in the vascular bundles of the nodules. The expression of these genes was repressed in the ineffective nodules, which had lower levels of nitrogenase activity.     

In vivo imaging of endoplasmic reticulum and distribution of mutant α-amylase in Aspergillus oryzae

Properly folded proteins destined for secretion exit through a specific subdomain of the endoplasmic reticulum (ER) known as transitional ER (tER) sites or ER exit sites (ERES). While such proteins in filamentous fungi localize at the hyphal tips overlapping the Spitzenk?rper, the distribution of misfolded proteins remains unknown. In the present study, we analyzed the distribution of mutant protein as well as ER and tER sites visualized by expression of AoClxA and AoSec13 fused with fluorescent protein, respectively, in the filamentous fungus Aspergillus oryzae. Discrete tER subdomains were visualized as the punctate dots of AoSec13 overlapping or associated with AoClxA distribution. Both ER and tER sites were concentrated near hyphal tips and formed apical gradients. Interestingly, while the expression of wild-type α-amylase fusion protein (AmyB-mDsRed) showed its localization coinciding with the Spitzenk?rper, a disulfide bond-deletion in AmyB causing its misfolding resulted in its accumulation in the subapical and basal ER, creating a reciprocal gradient to the tER sites. Furthermore, the reciprocal gradient enabled a clear distinction between the tER sites and the mutant AmyB accumulation sites near the apex. Based on these findings, we conclude that A. oryzae accumulates aberrant proteins toward basal hyphae while maintaining polarized tER sites for secretion of properly folded proteins at the hyphal tip.     

Identification of novel molecular candidates for fatty liver in the hyperlipidemic mouse model, HcB19

The inbred HcB19 mouse strain expresses a truncated form of thioredoxin interacting protein and is phenotypically characterized by fatty liver and elevated plasma triglycerides and VLDL. Recently, these mice have been proposed as an animal model for familial combined hyperlipidemia. The aim of the present study was identification of hepatic proteins specifically associated with the presence of fatty liver. Eighteen differential proteins were detected in whole-liver homogenate from HcB19, or the parental strain C3H, using 2D electrophoresis, and 11 of those were successfully identified by mass spectrometry. Five of the identified differential proteins were mitochondrial, two peroxisomal, two cytosolic, and two secretory. Four differential proteins were novel in the fatty liver proteome [i.e., aconitase, succinate dehydrogenase, propionyl CoA carboxylase alpha chain (PCCA), and 3-hydroxyanthranilate 3,4 dioxygenase (3HAAO)]. Of these, PCCA and 3HAAO are of particular interest because of their known functions in nicotinic acid metabolism (3HAAO) and ketogenesis (PCCA). We have newly identified several differential proteins in the hepatic proteome of mice with fatty liver, including PCCA and 3HAAO, and confirmed differential expression of previously reported proteins. These individual proteins, PCCA and 3HAAO, can be important in development of fatty liver or in the expression of hyperlipidemia.     

A proteomic survey of rat cerebral cortical synaptosomes

Previous findings from our laboratory and others indicate that two-dimensional gel electrophoresis (2-DE) can be used to study protein expression in defined brain regions, but mainly the proteins which are present in high abundance in glia are readily detected. The current study was undertaken to determine the protein profile in a synaptosomal subcellular fraction isolated from the cerebral cortex of the rat. Both 2-DE and liquid chromatography - tandem mass spectrometry (LC-MS/MS) procedures were used to isolate and identify proteins in the synaptosomal fraction and accordingly >900 proteins were detected using 2-DE; the 167 most intense gel spots were isolated and identified with matrix-assisted laser desorption/ionization - time of flight peptide mass fingerprinting or LC-MS/MS. In addition, over 200 proteins were separated and identified with the LC-MS/MS "shotgun proteomics" technique, some in post-translationally modified form. The following classes of proteins associated with synaptic function were detected: (a) proteins involved in synaptic vesicle trafficking-docking (e.g., SNAP-25, synapsin I and II, synaptotagmin I, II, and V, VAMP-2, syntaxin 1A and 1B, etc.); (b) proteins that function as transporters or receptors (e.g., excitatory amino acid transporters 1 and 2, GABA transporter 1); (c) proteins that are associated with the synaptic plasma membrane (e.g., post-synaptic density-95/synapse-associated protein-90 complex, neuromodulin (GAP-43), voltage-dependent anion-selective channel protein (VDACs), sodium-potassium ATPase subunits, alpha 2 spectrin, septin 7, etc.); and (d) proteins that mediate intracellular signaling cascades that modulate synaptic function (e.g., calmodulin, calcium-calmodulin-dependent protein kinase subunits, etc.). Other identified proteins are associated with mitochondrial or general cytosolic function. Of the two proteins identified as endoplasmic reticular, both interact with the synaptic SNARE complex to regulate vesicle trafficking. Taken together, these results suggest that the integrity of the synaptosomes was maintained during the isolation procedure and that this subcellular fractionation technique enables the enrichment of proteins associated with synaptic function. The results also suggest that this experimental approach can be used to study the differential expression of multiple proteins involved in alterations of synaptic function.     

Alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV

The incidence of hepatocellular carcinoma (HCC) is closely correlated with hepatitis B virus (HBV)-induced liver cirrhosis. Structural changes in the glycans of serum and tissue proteins are reliable indicators of liver damage. However, little is known about the alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV infection. This study compared the differential expression of liver glycopatterns in 7 sets of normal pericarcinomatous tissues (PCTs), cirrhotic, and tumor tissues from patients with liver cirrhosis and HCC induced by HBV using lectin microarrays. Fluorescence-based lectin histochemistry and lectin blotting were further utilized to validate and assess the expression and distribution of certain glycans in 9 sets of corresponding liver tissue sections. Eight lectins (e.g., Jacalin and AAL) revealed significant difference in cirrhotic tissues versus PCTs. Eleven lectins (e.g., EEL and SJA) showed significant alteration during cirrhotic and tumor progression. The expression of Galα1-3(Fucα1-2)Gal (EEL) and fucosyltransferase 1 was mainly increasing in the cytoplasm of hepatocytes during PCTs-cirrhotic-tumor tissues progression, while the expression of T antigen (ACA and PNA) was decreased sharply in cytoplasm of tumor hepatocytes. Understanding the precision alteration of liver glycopatterns related to the development of hepatitis, cirrhosis, and tumor induced by HBV infection may help elucidate the molecular mechanisms underlying the progression of chronic liver diseases and develop new antineoplastic therapeutic strategies.     

Differential expression of sarcoplasmic proteins in four heterogeneous ovine skeletal muscles

Fiber-type distribution is known to vary widely within and between muscles according to differences in muscle functions. 2-DE and MALDI-MS were used to investigate the molecular basis of muscle fiber type-related variability. We compared four lamb skeletal muscles with heterogeneous fiber-type composition that are relatively rich in fast-twitch fiber types, i.e., the semimembranosus, vastus medialis, longissimus dorsi, and tensor fasciae latae (TL). Our results clearly showed that none of the glycolytic metabolism enzymes detected, including TL which was most strongly glycolytic, made intermuscular differentiation possible. Muscle differentiation was based on the differential expression of proteins involved in oxidative metabolism, including not only citric acid cycle enzymes but also other classes of proteins with functions related to oxidative metabolism, oxidative stress, and probably to higher protein turnover. Detected proteins were involved in transport (carbonate dehydratase, myoglobin, fatty acid-binding protein), repair of misfolding damage (heat shock protein (HSP) 60 kDa, HSP-27 kDa, alpha-crystallin beta subunit, DJ1, stress-induced phosphoprotein), detoxification or degradation of impaired proteins (GST-Pi, aldehyde dehydrogenase, peroxiredoxin, ubiquitin), and protein synthesis (tRNA-synthetase). The fractionating method led to the detection of proteins involved in different functions related to oxidative metabolism that have not previously been shown concomitancy.     

脂代谢相关三基因突变小鼠肝脏差异表达蛋白组研究

应用双向电泳及质谱技术对5周龄三基因(apoE^-1- / LDLR^-1-/Lepr^db/db)联合突变小鼠和野生型小鼠肝组织的差异蛋白质进行比较研究,借此分析脂代谢相关三基因联合突变小鼠肝脏蛋白质表达特点,研究差异表达蛋白与血脂代谢紊乱和动脉粥样硬化的关系.在实验中检测到三基因联合突变小鼠和野生型小鼠肝脏中分别平均有（841±57）个和（1 017±50）个蛋白点（n=3）,两者的平均匹配率分别为71.9%,83.2%.三基因联合突变小鼠有140个蛋白点未能与野生型小鼠匹配,其中相差5倍以上的上调点和下调点分别为7个和39个.选取其中的6个点做质谱分析,鉴定为endoplasmin precursor(Grp-94)、酸性富亮氨酸核磷蛋白32家族成员A(acidic leucin-rich nuclear phosphoprotein 32 family member A)、转铁蛋白前体、果糖二磷酸酶1、纤维连接蛋白前体、补体C3前体,纤维蛋白原B β多肽7种蛋白. 该结果提示,差异表达的蛋白对三基因联合突变小鼠的血脂代谢紊乱和动脉粥样硬化发生发展过程起一定作用.     

Downregulation of diaphragm electron transport chain and glycolytic enzyme gene expression in sepsis.

Cellular energy metabolism is altered in sepsis as a consequence of dysfunction of mitochondrial electron transport and glycolytic pathways. The purpose of the present study was to determine whether sepsis is associated with compensatory increases in gene expression of electron transport chain and glycolytic pathway proteins or, alternatively, whether gene expression decreases in sepsis, contributing to abnormalities in energy metabolism. Studies were performed using diaphragms from control and endotoxin-treated (8 mg x kg(-1) x day(-1)) rats; at 48 h after endotoxin administration, animals were killed. Microarrays and RNAse protection assays were used to assess the expression of several electron transport chain components (cytochrome-c oxidase subunits Cox 5A, Cox 5B, and Cox 6A, ATP synthase, and ATP synthase subunit 5B) and of the rate-limiting enzyme for glycolysis, phosphofructokinase (PFK). Western blotting was used to assess protein levels for these electron transport chain subunits and PFK. Activity assays were used to assess electron transport chain and phosphofructokinase function. We found that sepsis evoked 1) a downregulation of genes encoding all examined electron transport chain components (e.g., cytochrome-c oxidase 5A decreased 45 + 7%, P < 0.01) and PFK (P < 0.001), 2) reductions in protein levels for these electron transport chain subunits and PFK (P < 0.05 for each), and 3) decreases in mitochondrial state 3 respiration rates and phosphofructokinase enzyme activity (P < 0.01 for each comparison). We speculate that these sepsis-induced reductions in the expression of genes encoding critical electron transport and glycolytic proteins contribute to the development and persistence of sepsis-induced abnormalities in cellular energy metabolism.     

Long-term adjustment of hepatic lipid metabolism after chronic stress and the role of FGF21

Chronic stress leads to post-traumatic stress disorder (PTSD) and metabolic disorders including fatty liver. We hypothesized that stress-induced molecular mechanisms alter energy metabolism, thereby promoting hepatic lipid accumulation even after a stress-free recovery period. In this context, we investigated fibroblast growth factor-21 (FGF21) as protective for energy and glucose homeostasis. FGF21 knockout mice (B6.129S6(SJL)-Fgf21tm1.2Djm; FGF21KO) and control mice (C57BL6; WT) were subjected to chronic variable stress. Mice were examined directly after acute intervention (Cvs) and long-term after 3 months of recovery (3mCvs). In WT, Cvs reduced insulin sensitivity and hepatic lipid accumulation, whilst fatty acid uptake increased. FGF21KO mice responded to Cvs with improved glucose tolerance, insulin resistance but liver triglycerides and plasma lipids were unaltered. Hepatic gene expression was specifically altered by genotype and stress e.g. by PPARa and SREBP-1 regulated genes. The stress-induced alteration of hepatic metabolism persisted after stress recovery. In hepatocytes at 3mCvs, differential gene regulation and secreted proteins indicated a genotype specific progression of liver dysfunction. Overall, at 3mCvs FGF21 was involved in maintaining mitochondrial activity, attenuating de novo lipogenesis, increased fatty acid uptake and histone acetyltransferase activity. Glucocorticoid release and binding to the FGF21 promoter may contribute to prolonged FGF21 release and protection against hepatic lipid accumulation. In conclusion, we showed that stress favors fatty liver disease and FGF21 protected against hepatic lipid accumulation after previous chronic stress loading by i) restored physiological function, ii) modulated gene expression via DNA-modifying enzymes, and iii) maintained energy metabolism.     

The molecular action of the novel insecticide, Pyridalyl

Pyridalyl is a recently discovered insecticide that exhibits high insecticidal activity against Lepidoptera and Thysanoptera. Pyridalyl action requires cytochrome P450 activity, possibly for production of a bioactive derivative, Pyridalyl metabolism being prevented by general P450 inhibitors. Apoptosis is apparently not involved in the cytotoxicity. Continuous culture of Spodoptera frugiperda Sf21 cells in sub-lethal doses of Pyridalyl, results in a Pyridalyl-resistant cell line. Probing the molecular action of Pyridalyl by comparison of the proteomes of Pyridalyl-resistant and -susceptible cell lines, revealed differential expression of a number of proteins, including the up-regulation of thiol peroxiredoxin (TPx), in the resistant cells. Treatment of Bombyx mori larvae with Pyridalyl, followed by comparison of the midgut microsomal sub-proteome, revealed the up-regulation of three proteasome subunits. Such subunits, together with Hsp70 stress proteins, glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) and thiol peroxiredoxin (TPx) were also up-regulated in the whole proteome of B. mori BM36 cells following treatment with the insecticide. The foregoing results lead to the hypothesis that cytochrome P450 action leads to an active Pyridalyl metabolite, which results in production of reactive oxygen species (ROS), that leads to damage to cellular macromolecules (e.g., proteins) and enhanced proteasome activity leads to increased protein degradation and necrotic cell death.     

Proteomic analysis of infiltrating ductal carcinoma tissues by coupled 2-D DIGE/MS/MS analysis

There is a growing interest in protein expression profiling aiming to identify novel diagnostic markers in breast cancer. Proteomic approaches such as two-dimensional differential gel electrophoresis coupled with tandem mass spectrometry analysis (2-D DIGE/MS/MS) have been used successfully for the identification of candidate biomarkers for screening, diagnosis, prognosis and monitoring of treatment response in various types of cancer. Identifying previously unknown proteins of potential clinical relevance will ultimately help in reaching effective ways to manage the disease. We analyzed breast cancer tissues from five tumor and five normal tissue samples from ten breast cancer subjects with infiltrating ductal carcinoma (IDC) by 2-D DIGE using two types of immobilized pH gradient (IPG) strips: pH 3-10 and pH 4-7. From all the spots detected, differentially expressed (p < 0.05 and ratio > 2) were 50 spots. Of these, 39 proteins were successfully identified by MS, representing 29 different proteins. Ten proteins were overexpressed in the tumor samples. The 2-D DIGE/MS/MS analysis revealed an increase in the expression levels in tumor samples of several proteins not previously associated with breast cancer, such as: macrophage-capping protein (CAPG), phosphomannomutase 2 (PMM2), ATPase ASN1, methylthioribose-1-phosphate isomerase (MRI1), peptidyl-prolyl cis-trans isomerase FKBP4, cellular retinoic acid-binding protein 2 (CRABP2), lamin B1 and keratin, type II cytoskeletal 8 (KRT8). Ingenuity Pathway Analysis (IPA) revealed highly significant (p = 10(-26)) interactions between the identified proteins and their association with cancer. These proteins are involved in many diverse pathways and have established roles in cellular metabolism. It remains the goal of future work to test the suitability of the identified proteins in samples of larger and independent patient groups.     

Differential Proteomic Analysis of Dimethylnitrosamine (DMN)‐Induced Liver Fibrosis

Liver fibrosis is a common pathological feature of many chronic liver diseases. To characterize the entire panorama of proteome changes in dimethylnitrosamine (DMN)‐induced liver fibrosis, isobaric tags for relative and absolute quantitation (iTRAQ)‐based differential proteomic analysis is performed with DMN‐induced liver fibrosis rats. A total of 4155 confidently identified proteins are found, with 365 proteins showing significant changes (fold changes of >1.5 or < 0.67, p < 0.05). In metabolic activation, proteins assigned to drug metabolism enzymes (e.g., CYP2D1) change, suggesting that the liver protection mechanism is activated to relieve DMN toxicity. In addition, the altered proteins of immune response and oxidative stress may activate hepatic stellate cells. Glucose metabolism disorder in DMN model rats is demonstrated by a decrease in key enzymes (e.g., ACSL1) in fatty acid metabolism, a tricabolic acid cycle‐related enzyme (SDH), glycogenolysis enzyme, and gluconeogenesis enzymes (PC, PCKGC) and by an increase in glycolysis enzymes (e.g., HXK1). Meanwhile, alterations in iron and calcium ion homeostasis proteins are observed. Our results also show that mitochondrial dysfunction may be involved in DMN hepatotoxicity. In conclusion, these altered liver proteins in the DMN model and control rats provide data for understanding the functional mechanism of liver fibrosis.     

Differential protein expression profile in the liver of pikeperch (Sander lucioperca) larvae fed with increasing levels of phospholipids

A comparative proteomic approach was used to assess the protein expression profile in the liver of 34 days old pikeperch larvae fed from day 10 post hatching, with three isoproteic and isolipidic formulated diets varying by their phospholipid (PL) contents (% dry diet weight): 1.4% (PL1), 4.7% (PL5) and 9.5% (PL9). Using 2D-DIGE minimal labelling of liver extracts, we were able to show 56 protein spots with a differential intensity (p < 0.05) depending on the dietary PL content. Among these spots, 11 proteins were unambiguously identified using nanoLC-MS/MS tandem mass spectrometry. In the PL9 larvae, our results indicate that the glycolytic pathway could be down-regulated due to the under-expression of the fructose biphosphate aldolase B and the phosphoglucomutase 1. Meanwhile, propionyl coenzyme A carboxylase (a gluconeogenic enzyme) was under-expressed. In addition, another gluconeogenic and lipogenic enzyme, pyruvate carboxylase, was identified in 3 different spots as being under-expressed in fish fed with the intermediate PL level (PL5). A high PL content increased the expression of sarcosine dehydrogenase, an enzyme involved in methionine metabolism, along with vinculin, a structural protein. Moreover, several stress proteins (glutathione S-transferase M, glucose regulated protein 75 and peroxiredoxin-1) were modulated in response to the dietary PL level and fatty acid composition. In the larvae fed with the lowest dietary PL content (PL1), over-expression of both GSTM and GRP75 might indicate a cellular stress in this experimental treatment, while the under-expression of Prx1 might indicate a lower defence against oxidative stress. In conclusion, this nutriproteomic approach showed significant modifications of protein expression in the liver of pikeperch larvae fed different PL contents, highlighting the importance of these nutrients and their influence on metabolism processes and on stress response.     

Comparative proteomics analysis of human stem cells from dental gingival and periodontal ligament

Dental stem cells isolated from oral tissues have been shown to provide with high proliferation ability and multilineage differentiation potential. Gingival mesenchymal stem cells (GMSCs) and periodontal ligament stem cells (PDLSCs), kinds of dental stem cells, can be used as substitutes for tissue repair materials because of their similar regenerative functions. In this study, we aim to explore the similarities and differences between the protein profiles of GMSCs and PDLSCs through quantitative proteomics. A total of 2821 proteins were identified and retrieved, of which 271 were upregulated and 57 were downregulated in GMSCs compared to PDLSCs. Gene Ontology (GO) analysis demonstrated that the 328 differentially abundant proteins (DAPs) were involved in the regulation of gene expression, metabolism, and signal transduction in biological process, mainly distributed in organelles related to vesicle transport, and involved in the molecular function of binding protein. And Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DAPs were committed to regulating the synthesis of proteasome and spliceosome. Real-time quantitative polymerase chain reaction (RT-qPCR) results showed that ARPC1B, PDAP1, and SEC61B can be used as special markers to distinguish GMSCs from PDLSCs. This research contributes to explaining the molecular mechanism and promoting the clinical application of tissue regeneration of GMSCs and PDLSCs.     

Postnatal Hyperoxia Exposure Differentially Affects Hepatocytes and Liver Haemopoietic Cells in Newborn Rats

Premature newborns are frequently exposed to hyperoxic conditions and experimental data indicate modulation of liver metabolism by hyperoxia in the first postnatal period. Conversely, nothing is known about possible modulation of growth factors and signaling molecules involved in other hyperoxic responses and no data are available about the effects of hyperoxia in postnatal liver haematopoiesis. The aim of the study was to analyse the effects of hyperoxia in the liver tissue (hepatocytes and haemopoietic cells) and to investigate possible changes in the expression of Vascular Endothelial Growth Factor (VEGF), Matrix Metalloproteinase 9 (MMP-9), Hypoxia-Inducible Factor-1α (HIF-1α), endothelial Nitric Oxide Synthase (eNOS), and Nuclear Factor-kB (NF-kB). Experimental design of the study involved exposure of newborn rats to room air (controls), 60% O₂ (moderate hyperoxia), or 95% O₂ (severe hyperoxia) for the first two postnatal weeks. Immunohistochemical and Western blot analyses were performed. Severe hyperoxia increased hepatocyte apoptosis and MMP-9 expression and decreased VEGF expression. Reduced content in reticular fibers was found in moderate and severe hyperoxia. Some other changes were specifically produced in hepatocytes by moderate hyperoxia, i.e., upregulation of HIF-1α and downregulation of eNOS and NF-kB. Postnatal severe hyperoxia exposure increased liver haemopoiesis and upregulated the expression of VEGF (both moderate and severe hyperoxia) and eNOS (severe hyperoxia) in haemopoietic cells. In conclusion, our study showed different effects of hyperoxia on hepatocytes and haemopoietic cells and differential involvement of the above factors. The involvement of VEGF and eNOS in the liver haemopoietic response to hyperoxia may be hypothesized.     

Proteomic analysis of tumor establishment and growth in the B16-F10 mouse melanoma model

The B16-F10 mouse model of melanoma is a widely used model to study many aspects of cancer biology and therapeutics in a solid tumor. Melanomas aggressively progress within a dynamic microenvironment containing in addition to tumor cells, stroma cells and components such as fibroblasts, immune cells, vascular cells, extracellular matrix (ECM) and extracellular molecules. The goal of this study was to elucidate the processes of tumor progression by identifying differentially expressed proteins in the tumor mass during specific stages of tumor growth. A comparative proteome analysis was performed on B16-F10 derived tumors in C57BL/6 mice at days 3, 5, 7, and 10. Statistical approaches were used to determine quantitative differential protein expression at each tumor time stage. Hierarchical clustering of 44 protein spots (p < 0.01) revealed a progressive change in the tumor mass when all 4 time stages were classified together, but there was a clear switch in expression of these proteins between the day 5 and the day 7 tumors. A trend analysis showed 53 protein spots (p < 0.001) following 6 predominant kinetic paths of expression as the tumor progressed. The protein spots were then identified using MALDI-TOF mass spectrometry. Proteins involved in glycolysis, inflammation, wounding, superoxide metabolism, and chemotaxis increased during tumorigenesis. From day 3 to day 7 VEGF and active cathepsin D were induced 7-fold and 4-fold, respectively. Proteins involved in electron transport, protein folding, blood coagulation, and transport decreased during tumorigenesis. This work illustrates changes in the biology of the B16-F10 tumor mass during tumor progression.     

Cells lacking IKKα show nuclear cyclin D1 overexpression and a neoplastic phenotype: role of IKKα as a tumor suppressor

The catalytic subunits of IκB kinase (IKK) complex, IKKα and IKKβ, are involved in activation of NF-κB and in mediating a variety of other biological functions. Though these proteins have a high-sequence homology, IKKα exhibits different functional characteristics as compared with IKKβ. Earlier, we have shown that cyclin D1 is overexpressed and predominantly localized in the nucleus of IKKα(-/-) cells, indicating that IKKα regulates turnover and subcellular distribution of cyclin D1, which is mediated by IKKα-induced phosphorylation of cyclin D1. Because cyclin D nuclear localization is implicated in tumor development, we examined whether the absence of IKKα leads to tumor development as well. In the current study, we show that IKKα plays a critical role in tumorigenesis. Though IKKα(-/-) MEF cells show a slower anchorage-dependent growth, they are clonogenic in soft agar. These cells are tumorigenic in nude mice. Microarray analysis of IKKα(-/-) cells indicates a differential expression of genes involved in proliferation and apoptosis. Furthermore, analysis of microarray data of human lung cancer cell lines revealed decreased IKKα RNA expression level as compared with cell lines derived from normal bronchial epithelium. These results suggest that IKKα may function as a tumor suppressor gene. Absence of IKKα may induce tumorigenicity by nuclear localization of cyclin D1 and modulating the expression of genes involved in neoplastic transformation.