Vector Control Lines Research Articles

Abstract 2338: Molecular mechanisms underlying cellular transformation by Tumor protein D52 overexpression

Abstract Background: Tumor protein D52 (D52) overexpression is characteristic of both adult and childhood cancers and has been associated with reduced overall survival in breast cancer patients and an increase in proliferation and metastatic capacity of transfected cell lines. However, little is known about the mechanism(s) by which D52 overexpression contributes to cellular transformation or tumour progression. Aims: We aimed to identify genes which are differentially expressed in response to D52 expression in 3T3 fibroblasts, and to use these gene expression changes to predict cellular phenotypes which are altered by D52 expression. Methods: Gene expression profiling was performed using Affymetrix expression microarray analysis. Up- and down-regulated gene lists were analysed using GOSTAT, Metacore and literature searches. Differential expression of candidate proteins were validated using Western blot analysis of intracellular protein extracts or precipitates from conditioned media. Migration and invasion assays were performed using Boyden chambers. Results: D52 expression was associated with over 300 genes being differentially expressed at least two-fold relative to vector controls. A number of overexpressed genes were found to be normally involved in the inflammatory response. We focused on validating candidates such as COX-2, MMP-2 and Osteopontin as being overexpressed in D52-transfected cells relative to vector controls. We also confirmed an upregulation of phospho-AKT in D52 expressing cells, as reported by others. GOSTAT analysis revealed that gene ontology terms related to the extracellular matrix (ECM) were most over-represented amongst differentially expressed genes. The implied resultant ECM re-modelling was examined through migration and invasion assays, in which D52 expressing cells showed a significantly increased migratory and invasive capacity relative to vector control lines. Conclusions: These results implicate D52 as a pro-inflammatory intermediate, whose expression may act to drive cancer progression via an increase in phospho-AKT, followed by the production of inflammatory molecules such as Osteopontin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2338.

Transgenic expression of fern Pteris vittata glutaredoxin PvGrx5 in Arabidopsis thaliana increases plant tolerance to high temperature stress and reduces oxidative damage to proteins

A glutaredoxin of the fern Pteris vittata PvGRX5 was previously implicated in arsenic tolerance. Because of possible involvements of glutaredoxins in metabolic adaptations to high temperature stress, transgenic Arabidopsis lines constitutively expressing PvGRX5 were evaluated for thermotolerance. Homozygous lines expressing PvGRX5 exhibited significantly greater tolerance to high temperature stress than the vector control and wild-type, based upon growth during stress and during recovery from stress, and this was related to leaf glutaredoxin specific activities. Measurements of tissue ion leakage, thiobarbituric acid reactive substances and protein carbonyl content showed that PvGRX5-expressors were significantly (P < 0.05) less affected by the high temperature treatment compared to wild-type and vector control lines for damage to membranes and proteins. Immunoblots indicated that specific protein bands, carbonylated during the stress treatment in the control lines, were protected in PvGRX5-expressors, thus implicating PvGRX5 in heat tolerance, likely mediated through cellular protection against oxidative stress.

Expression of a Pteris vittata glutaredoxin PvGRX5 in transgenic Arabidopsis thaliana increases plant arsenic tolerance and decreases arsenic accumulation in the leaves

Chinese brake fern Pteris vittata hyperaccumulates arsenic in its fronds. In a study to identify brake fern cDNAs in arsenic resistance, we implicated a glutaredoxin, PvGRX5, because when expressed in Escherichia coli, it improved arsenic tolerance in recombinant bacteria. Here, we asked whether PvGRX5 transgenic expression would alter plant arsenic tolerance and metabolism. Two lines of Arabidopsis thaliana constitutively expressing PvGrx5 cDNA were compared with vector control and wild-type lines. PvGRX5-expressors were significantly more tolerant to arsenic compared with control lines based on germination, root growth and whole plant growth under imposed arsenic stress. PvGRX5-expressors contained significantly lower total arsenic compared with control lines following treatment with arsenate. Additionally, PvGRX5-expressors were significantly more efficient in their arsenate reduction in vivo. Together, our results indicate that PvGRX5 has a role in arsenic tolerance via improving arsenate reduction and regulating cellular arsenic levels. Paradoxically, our results suggest that PvGRX5 from the arsenic hyperaccumulator fern can be used in a novel biotechnological solution to decrease arsenic in crops.

A Cation-regulated and Proton Gradient-dependent Cation Transporter from Chlamydomonas reinhardtii Has a Role in Calcium and Sodium Homeostasis

The CrCAX1 gene encoding a Ca2+/H+ and Na+/H+ exchanger was cloned and characterized from the unicellular green alga Chlamydomonas reinhardtii to begin to understand the mechanisms of cation homeostasis in this model organism. CrCAX1 was more closely related to fungal cation exchanger (CAX) genes than those from higher plants but has structural characteristics similar to plant Ca2+/H+ exchangers including a long N-terminal tail. When CrCAX1-GFP was expressed in Saccharomyces cerevisiae, it localized at the vacuole. CrCAX1 could suppress the Ca2+-hypersensitive phenotype of a yeast mutant and mediated proton gradient-dependent Ca2+/H+ exchange activity in vacuolar membrane vesicles. Ca2+ transport activity was increased following N-terminal truncation of CrCAX1, suggesting the existence of an N-terminal auto-regulatory mechanism. CrCAX1 could also provide tolerance to Na+ stress when expressed in yeast or Arabidopsis thaliana because of Na+/H+ exchange activity. This Na+/H+ exchange activity was not regulated by the N terminus of the CrCAX1 protein. A subtle tolerance by CrCAX1 in yeast to Co2+ stress was also observed. CrCAX1 was transcriptionally regulated in Chlamydomonas cells grown in elevated Ca2+ or Na+. This study has thus uncovered a novel eukaryotic proton-coupled transporter, CrCAX1, that can transport both monovalent and divalent cations and that appears to play a role in cellular cation homeostasis by the transport of Ca2+ and Na+ into the vacuole.

The Effects of Cyclooxygenase-2 Expression in Prostate Cancer Cells: Modulation of Response to Cytotoxic Agents

Cyclooxygenase (COX)-2 has emerged as an exciting target for therapeutic intervention in the management of cancer. Immunohistochemistry studies have indicated higher expression of COX-2 in cancerous versus benign prostatic tissue. We have explored the role of COX-2 in prostate cancer in terms of attenuation of apoptosis and sensitivity to pharmacological agents, including COX-2 inhibitors. The human prostate cancer cell line LNCaP was stably transfected with COX-2 (LNCaPCOX-2) and compared with the empty vector control line (LNCaPneo). Chemosensitivity testing indicated no change in sensitivity to the cytotoxic effects of COX-2 inhibitors celecoxib or sulindac or VP16. However, LNCaPCOX-2 cells showed 3-fold resistance to carboplatin, which was partially reversed by coincubation with the phosphatidylinositol 3-kinase inhibitor wortmannin. Concomitant with reduced apoptotic response to cytotoxic agents, LNCaPCOX-2 cells expressed increased levels of survivin and Bcl-2 with enhanced activation of AKT. We also investigated the effects of celecoxib on expression levels of genes relevant to prostate cancer and drug resistance in our model system using quantitative polymerase chain reaction analysis. Celecoxib treatment resulted in highly significant increases in the mRNA expression of the smooth muscle component desmin, the detoxification enzyme glutathione S-transferase pi (GSTpi), and nonsteroidal anti-inflammatory response gene (NAG-1) in the LNCaPCOX-2 cell line compared with LNCaPneo cells. Significant decreases in survivin levels and increases in GSTpi and NAG-1 appeared to be COX-2-dependent effects because they were more pronounced in LNCaPCOX-2 cells. Our findings indicate both COX-2-dependent and -independent mechanisms attributable to celecoxib and support its utility in the management of prostate cancer.

HGF Mediates Cell Proliferation of Human Mesothelioma Cells through a PI3K/MEK5/Fra-1 Pathway

The ligand hepatocyte growth factor/scatter factor (HGF) and its receptor tyrosine kinase, c-Met, are highly expressed in most human malignant mesotheliomas (MMs) and may contribute to their increased growth and viability. Based upon our observation that RNA silencing of fos-related antigen 1 (Fra-1) inhibited c-met expression in rat mesotheliomas (1), we hypothesized that Fra-1 was a key player in HGF-induced proliferation in human MMs. In three of seven human MM lines evaluated, HGF increased Fra-1 levels and phosphorylation of both extracellular signal-regulated kinase 5 (ERK5) and AKT that were inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor, LY290042. HGF-dependent phosphorylation and Fra-1 expression were decreased after knockdown of Fra-1, whereas overexpression of Fra-1 blocked the expression of mitogen/extracellular signal-regulated kinase kinases (MEK)5 at the mRNA and protein levels. Stable MM cell lines using a dnMEK5 showed that basal Fra-1 levels were increased in comparison to empty vector control lines. HGF also caused increased MM cell viability and proliferating cell nuclear antigen (PCNA) expression that were abolished by knockdown of MEK5 or Fra-1. Data suggest that HGF-induced effects in some MM cells are mediated via activation of a novel PI3K/ERK5/Fra-1 feedback pathway that might explain tumor-specific effects of c-Met inhibitors on MM and other tumors.

Soluble CD44 secretion contributes to the acquisition of aggressive tumor phenotype in human colon cancer cells

CD44, a widely distributed cell surface glycoprotein and a receptor for hyaluronan (HA), has been implicated in facilitating tumor growth and metastasis, antiapoptosis and directional motility of cancer cells. In order to investigate the role of soluble CD44 (CD44 sol) in colon cancer cell growth, SW620, a human colon cancer cell line deficient in CD44 expression was stably transfected with human CD44 cDNA containing exons 1–5, 15 and 16 of the human CD44. Western blot analyses demonstrated the presence of 78 kDa soluble CD44 protein in the culture supernatant of stably transfected cell lines (CD44 sol clones) and were not detected in the empty vector control line (clone m). The CD44 sol transfected cells showed higher cell proliferation and clonal growth in vitro, confirmed by MTT and clonogenic assays respectively, when compared to the control cells. Cell adhesion to hyaluronan was significantly lower with CD44 sol cells compared to the control cells. Western blot analyses were negative for cleaved PARP in lysates from CD44 sol cells, suggesting resistance to apoptosis. These findings indicate that the secretion of soluble CD44 contributes to colon cancer growth in vitro, possibly as a decoy receptor.

Gastrodia anti-fungal protein from the orchid Gastrodia elata confers disease resistance to root pathogens in transgenic tobacco

Diseases of agricultural crops are caused by pathogens from several higher-order phylogenetic lineages including fungi, straminipila, eubacteria, and metazoa. These pathogens are commonly managed with pesticides due to the lack of broad-spectrum host resistance. Gastrodia anti-fungal protein (GAFP; gastrodianin) may provide a level of broad-spectrum resistance due to its documented anti-fungal activity in vitro and structural similarity to insecticidal lectins. We transformed tobacco (Nicotiana tabacum cv. Wisconsin 38) with GAFP-1 and challenged transformants with agriculturally important plant pathogens from several higher-order lineages including Rhizoctonia solani (fungus), Phytophthora nicotianae (straminipile), Ralstonia solanacearum (eubacterium), and Meloidogyne incognita (metazoan). Quantitative real-time PCR and western blotting analysis indicated that GAFP-1 was transcribed and translated in transgenic lines. When challenged by R. solani and P. nicotianae, GAFP-1 expressing lines had reduced symptom development and improved plant vigor compared to non-transformed and empty vector control lines. These lines also exhibited reduced root galling when challenged by M. incognita. Against R. solanacearum expression of GAFP-1 neither conferred resistance, nor exacerbated disease development. These results indicate that heterologous expression of GAFP-1 can confer enhanced resistance to a diverse set of plant pathogens and may be a good candidate gene for the development of transgenic, root-disease-resistant crops.

Advanced Glycation End Products Inhibit Tubulogenesis and Migration of Kidney Epithelial Cells in an Ezrin-Dependent Manner

Nonenzymatic glycation of proteins to form advanced glycation end products (AGE) is implicated in diabetic complications, including nephropathy. It was shown recently that AGE bind to the ERM (ezrin, radixin, and moesin) family of membrane-cytoskeletal linker proteins in renal homogenates. Herein is reported the effects of AGE-BSA on ezrin-dependent LLC-PK1 kidney epithelial cellular functions: migration and hepatocyte growth factor (HGF)-induced tubulogenesis. LLC-PK1 cells were stably transfected with cDNA for ezrin sense, ezrin antisense, and N-ezrin. Transfection of LLC-PK1 cells with ezrin antisense and dominant negative N-ezrin decreased basal tubulogenesis and migration relative to vector-only transfection, establishing the ezrin dependency of these processes. AGE-BSA (20 or 40 microM) significantly decreased HGF-induced tubulogenesis and basal migration in two vector control lines relative to BSA-treated cells. However, AGE-BSA inhibition of both HGF-induced tubulogenesis and migration was overcome by overexpressing ezrin. These results demonstrate that the AGE-ezrin interaction significantly alters cellular function. These changes may be relevant to detrimental renal consequences as a result of diabetes.

The chemokine GRO-α (CXCL1) confers increased tumorigenicity to glioma cells

The chemokine GRO-alpha (CXCL1) has been found to mediate the proliferation of glia progenitor cells during neural development. As malignant gliomas are thought to arise from glia progenitors or their differentiated counterparts, astrocytes or oligodendrocytes, we have investigated whether GRO-alpha regulates the tumor characteristics of glioma cells. We found first that resected glioma specimens were strongly immunoreactive for GRO-alpha expression in cells with the morphology of tumor cells. In culture, the U251 glioma line transfected to overexpress GRO-alpha had elevated levels of motility and invasiveness. GRO-alpha transfectants increased their expression of several proteins associated with migratory behavior, including matrix metalloproteinase-2, beta1-integrin and SPARC. The implantation of GRO-alpha glioma clones into the brain of nude mice caused the early demise of mice and this was associated with the formation of larger intracerebral tumors when compared with mice implanted with vector control lines. These results implicate GRO-alpha in gliomas and suggest that the dysregulation of a glia proliferative factor contributes to tumorigenesis. Targeting GRO-alpha may be a useful therapeutic tool to control brain tumor biology.

OsCDPK13, a calcium-dependent protein kinase gene from rice, is induced by cold and gibberellin in rice leaf sheath

Calcium-dependent protein kinases (CDPKs) play an important role in rice signal transduction, but the precise role of each individual CDPK is still largely unknown. Recently, a full-length cDNA encoding OsCDPK13 from rice seedling was isolated. To characterize the function of OsCDPK13, its responses to various stresses and hormones were analyzed in this study. OsCDPK13 accumulated in 2-week-old leaf sheath and callus, and became phosphorylated in response to cold and gibberellin (GA). OsCDPK13 gene expression and protein accumulation were up-regulated in response to GA3 treatment, but suppressed in response to abscisic acid and brassinolide. Antisense OsCDPK13 transgenic rice lines were shorter than the vector control lines, and the expression of OsCDPK13 was lower in dwarf mutants of rice than in wild type. Furthermore, OsCDPK13 gene expression and protein accumulation were enhanced in response to cold, but suppressed under salt and drought stresses. Sense OsCDPK13 transgenic rice lines had higher recovery rates after cold stress than vector control rice. The expression of OsCDPK13 was stronger in cold-tolerant rice varieties than in cold-sensitive ones. The results suggest that OsCDPK13 might be an important signaling component in the response of rice to GA and cold stress.

Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca2+-dependent potassium channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein has the ability to function as both a chloride channel and a channel regulator. The loss of these functions explains many of the manifestations of the cystic fibrosis disease (CF), including lung and pancreatic failure, meconium ileus, and male infertility. CFTR has previously been implicated in the cell regulatory volume decrease (RVD) response after hypotonic shocks in murine small intestine crypts, an effect associated to the dysfunction of an unknown swelling-activated potassium conductance. In the present study, we investigated the RVD response in human tracheal CF epithelium and the nature of the volume-sensitive potassium channel affected. Neither the human tracheal cell line CFT1, expressing the mutant CFTR-DeltaF508 gene, nor the isogenic vector control line CFT1-LC3, engineered to express the betagal gene, showed RVD. On the other hand, the cell line CFT1-LCFSN, engineered to express the wild-type CFTR gene, presented a full RVD. Patch-clamp studies of swelling-activated potassium currents in the three cell lines revealed that all of them possess a potassium current with the biophysical and pharmacological fingerprints of the intermediate conductance Ca(2+)-dependent potassium channel (IK, also known as KCNN4). However, only CFT1-LCFSN cells showed an increase in IK currents in response to hypotonic challenges. Although the identification of the molecular mechanism relating CFTR to the hIK channel remains to be solved, these data offer new evidence on the complex integration of CFTR in the cells where it is expressed.

Increased Resistance to Apoptosis in Cells Overexpressing Thymosin Beta Four: A Role for Focal Adhesion Kinase pp125FAK

Loss of adherence to substrate can, by itself, induce apoptosis (anoikis) in epithelial cells, but does not do so in fibroblasts. To test the idea that adherence transmits signals that inhibit apoptosis even in fibroblasts, we took advantage of the greatly increased adherence to the substratum observed in NIH3T3 cell lines that overexpress thymosin beta four. We treated overexpressing (OE) and vector control lines with either ultraviolet light (UV) or tumor necrosis factor alpha (TNF alpha). When the cells were on a substratum, the more adherent OE cells were 2-fold more resistant to apoptosis induced by either treatment than vector controls. In contrast, when the cells were treated with either agent while in suspension, the difference in resistance between OE cells and vector controls was lost. Thus the increased resistance to apoptosis was dependent on adherence. There was no difference in the content of bcl-2 in the OE cells vs the controls. A connection between pp125FAK and resistance to apoptosis has been previously shown in primary cultures of fibroblasts. The Tbeta4 overexpressing cells have approximately 1.4x more pp125FAK than the controls, and the kinase is approximately 2-fold more phosphorylated in adherent OE cells than in the vector controls. The phosphorylation of pp125FAK decreased strikingly when the cells were put into suspension. In addition, twice as much paxillin associated with pp125FAK in OE adherent cells as in vector controls, but this difference was also lost in suspended cells. Our results support the concept of an adherence dependent pp125FAK-paxillin signalling pathway in fibroblasts that inhibits damage-induced apoptosis.

Reduced interleukin-8 production by cystic fibrosis airway epithelial cells.

The acquisition of Pseudomonas aeruginosa in the airways of patients with cystic fibrosis (CF) is the initial event leading to bronchiectasis and lung disease. Although the host factors that permit initial airway colonization are largely unknown, recent studies suggest that secretion of interleukin (IL)-8 by airway epithelia and local recruitment of neutrophils is the final pathway in a pulmonary cytokine network. To determine whether differences in cytokine production exist between normal and CF airway epithelia, secretion of immunoreactive IL-8 and IL-10 as well as specific messenger RNA (mRNA) abundance were compared in airway epithelia expressing normal and mutant CF transmembrane regulator. After induction with IL-1beta, a CF airway cell line engineered to express the wild-type CF gene (CFT1-LCFSN) secreted significantly more immunoreactive IL-8 than did its isogenic parent that expressed the mutant CF gene (CFT1) or an isogenic vector control line (CFT1-LC3). Further studies with the three related cell lines demonstrated that expression of CFT1-LCFSN was associated with a significant increase in uninduced secretion of immunoreactive IL-8 as well as a 10- to 20-fold increase in IL-8 mRNA abundance when compared with the isogenic lines expressing the mutant gene. IL-1beta induction and intracellular accumulation of IL-8 appeared to be unaffected by CF genotype. These studies suggest that IL-8 secretion by CF airway epithelial cells is defective and may contribute to Pseudomonas persistence in the CF airway. Further studies are needed to confirm this difference in other cell lines and determine the linkage between IL-8 production and CF gene expression.

Phosphorylation of ethanolamine, methylethanolamine, and dimethylethanolamine by overexpressed ethanolamine kinase in NIH 3T3 cells decreases the co-mitogenic effects of ethanolamines and promotes cell survival.

Ethanolamine (Etn), as well as its N-methyl (MeEtn) and N,N-dimethyl (Me2Etn) analogues, were recently shown to potentiate the stimulatory effect of insulin on DNA synthesis in serum-starved NIH 3T3 fibroblasts. In the present work we assessed the impact of the co-mitogenic effects of Etn and its methyl analogues on cell proliferation and cell survival, and examined whether the cell growth regulatory effects of these ethanolamines involve an Etn-kinase-mediated phosphorylation step. For this purpose, NIH 3T3 sublines highly overexpressing Drosophila Etn kinase and an appropriate vector control line were utilized and the effects of Etn, MeEtn, Me2Etn, methylamine (MeNH2), and dimethylamine (Me2NH) were studied. 31P-NMR analysis of the water-soluble cell metabolites revealed that both MeEtn and Me2Etn, but not choline, are excellent substrates for the expressed Etn kinase. The methylated ethanolamines (MeEtn and Me2Etn) and methylamines (MeNH2, Me2NH) were used as Etn models that can or cannot be phosphorylated, respectively. In serum-starved vector control cells, both MeNH2 (1 mM) and Me2NH (1 mM) were more effective than Etn in enhancing insulin-induced DNA synthesis, and both were almost as effective as MeEtn and Me2Etn. However, in the Etn kinase overexpressor cells the potentiating effects of Etn, MeEtn and Me2Etn, but not those of MeNH2 and Me2NH, were significantly reduced. Moreover, in the overexpressor cells, lower concentrations of Etn (50-200 microM) inhibited the combined mitogenic effects of Me2NH (1 mM) and insulin. These data are consistent with a mechanism in which the phosphorylated and non-phosphorylated ethanolamines are negative and positive regulators of insulin-induced mitogenesis, respectively. After incubating the cells for 13 days in serum-free medium in 96-well microplates, there was a steady decrease in cell numbers in both cell lines. However, between 6-13 days, 0.1-1 mM MeEtn and, particularly, Me2Etn provided significant protection against cell death in the Etn kinase overexpressor cells. In vector control cells, only Me2Etn in combination with insulin had similar effects on cell survival. The data suggest that phosphorylated ethanolamines may function as promoters of cell survival.

Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants.

Expression of the tobacco mosaic virus 30-kD movement protein (TMV MP) gene in tobacco plants increases the plasmodesmatal size exclusion limit (SEL) 10-fold between mesophyll cells in mature leaves. In the present study, we examined the structure of plasmodesmata as a function of leaf development. In young leaves of 30-kD TMV MP transgenic (line 274) and vector control (line 306) plants, almost all plasmodesmata were primary in nature. In both plant lines, secondary plasmodesmata were formed, in a basipetal pattern, as the leaves underwent expansion growth. Ultrastructural and immunolabeling studies demonstrated that in line 274 the TMV MP accumulated predominantly in secondary plasmodesmata of nonvascular tissues and was associated with a filamentous material. A developmental progression was detected in terms of the presence of TMV MP; all secondary plasmodesmata in the tip of the fourth leaf contained TMV MP in association with the filamentous material. Dye-coupling experiments demonstrated that the TMV MP-induced increase in plasmodesmatal SEL could be routinely detected in the tip of the fourth leaf, but was restricted to mesophyll and bundle sheath cells. These findings are discussed with respect to the structure and function of plasmodesmata, particularly those aspects related to virus movement.