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Gabriela Aust

Publications and source records attributed to Gabriela Aust.

18 recordsLinked to original sources
PubMed2006

Expression patterns and novel splicing variants of glutathione-S-transferase isoenzymes of human lung and hepatocyte cell lines.

Polymorphic glutathione S-transferase (GST) enzymes are involved in the metabolism of xenobiotics. They are of particular interest when studying disease susceptibility and adverse drug responses. The present work deals with the genetic polymorphisms and expression of the five GST classes (alpha, mu, pi, theta and zeta) in human lung and hepatocyte cell lines. We have determined their bioavailability for in vitro approaches. Common genetic polymorphisms of GSTM1 (*0, null), GSTT1 (*0) and GSTP1 (*A/*B, I105V) are detectable. The frequencies of the polymorphisms are within the expected range for a Caucasian population with one exception. The GSTM1*0 allele is 1.5-fold more frequent in lung cell lines. GST mRNAs are frequently but not uniformly distributed among unstimulated in vitro conditions. Lung cell lines show an approximately six-fold higher total GST transcript expression than hepatocyte cell lines. Additional GST transcripts have been identified for GSTT1; they represent alternative new splicing variants that occur in cancerous cell lines and in healthy lung tissue and blood. GST enzyme activity is mainly influenced by GSTP1. The activity promoted by 1-chloro-2,4-dinitrobenzene is significantly correlated to the GSTP1 mRNA expression level (R2=0.77, P<0.001). Individual human cell lines thus express GST isoenzymes in a similar pattern to human tissue. The most common genetic polymorphisms are present among the cell lines and have to be considered for in vitro stimulation approaches in a combinatory pattern.

Alleles↗
PubMed2006

Diversity of CD97 in smooth muscle cells.

CD97, an epidermal growth factor (EGF)-TM7 receptor, is not restricted to hematopoetic and carcinoma cells but is also found on smooth muscle cells (SMC). We have examined its location and biochemical structure in various normal and tumorigenic SMC-containing tissues. SMC of the urinary bladder, lung bronchi and bronchioles, myometrium, and gastrointestinal tract were immunohistologically stained by using monoclonal antibodies (mabs) to the CD97 stalk region (CD97(stalk)). Mabs directed against an N-glycosylation-dependent epitope within the EGF-domains (CD97(EGF)) did not bind to normal SMC. Vascular SMC, which was also CD97(EGF)-negative, showed further CD97 heterogeneity. Only a few, if any, SMC from the aorta or elastic arteries of the systemic circulation were positive for CD97 mRNA and therefore also for CD97(stalk). CD97(stalk)-positive SMC were slightly more numerous in muscular and peripheral arteries. In contrast, most venous SMC expressed CD97(stalk). A comparison with other SMC molecules revealed a similar but not identical staining pattern for CD97(stalk) and desmin. Further CD97 heterogeneity was observed during SMC transformation. All leiomyomas (n=5) and nine out of 21 leiomyosarcomas were positive for both CD97(stalk) and CD97(EGF). As expected, CD97(EGF)-positive SMC tumors expressed partly N-glycosylated CD97. Seven out of 21 leiomyosarcomas were completely devoid of CD97. Thus, CD97 showed variable expression in vascular and biochemical modification in tumorigenic SMC, suggesting that the function of the molecule is specific for the SMC subtype.

Adult↗
PubMed2006

Intraoperative subcutaneous or intrasplenic vaccination with modified autologous tumor cells leads to enhanced survival in a mouse tumor model.

PURPOSE: We investigated the effect of intraoperative intrasplenic or subcutaneous vaccination with modified tumor cells on tumor progression in a mouse model. METHODS: Pre-established B16 melanomas on C57/Bl6 mice were surgically removed; mice were vaccinated intraoperatively with B16 cells transfected with an IL-12-encoding pRSC construct, the empty plasmid, or B16 frozen cells. Cells were given either intrasplenically or subcutaneously. Intrasplenic effects of vaccination were examined along with survival data. Mice without tumor recurrence underwent a second tumor implantation. RESULTS: Animals administered IL-12 pRSC cells showed significant alterations in the spleen, such as higher percentages of (activated) CD4+ and CD8+ T cells and tumor-specific CD4+ T cells among splenocytes. The tumor recurrence rate after resection ranged from 13 to 36%. Cases with recurrent tumors in particular benefited in all therapy groups, resulting in enhanced (tumor-free) survival, reduced tumor growth and lower metastasis rates. Following macroscopic complete tumor resection, the optimum outcome resulted from vaccination with IL-12 pRSC cells into the spleen and subcutaneously administered frozen cells. Survival times were enhanced in all therapy groups after tumor reimplantation, although results were not significant. CONCLUSIONS: Intraoperative whole-cell vaccination with autologous tumor cells yields promising data, and could be considered as a future option in adjuvant cancer therapy.

Animals↗
PubMed2006

CD97 overexpression in tumor cells at the invasion front in colorectal cancer (CC) is independently regulated of the canonical Wnt pathway.

Invasion of colorectal carcinomas (CC) is characterized by nuclear accumulation of beta-catenin, a key component of the Wnt pathway, in scattered tumor cells. beta-catenin acts in cooperation with T-cell factor (Tcf) HMG-box transcription factors as a transcriptional activator of genes involved in tumor progression. Overexpression of CD97, a molecule that correlates with dedifferentiation and tumor stage in CC, parallels to nuclear translocation of beta-catenin. Here, we investigated whether CD97 is a direct beta-catenin/Tcf target gene. SW480 CC cells were used to mimic the phenotypical switch between central and invasive tumor areas. We demonstrate two-fold higher CD97 expression and nuclear beta-catenin accumulation in cells grown at low density compared to cells cultured at high density, showing membrane-associated beta-catenin. This suggests that CD97 expression correlates with the cellular localization of beta-catenin. However, CD97 mRNA expression levels were not affected by Tcf-1 or Tcf-4 as determined in inducible dominant-negative (dn) Tcf CC cell lines. Furthermore, co-expression of wildtype (wt) or S33A mutated beta-catenin with Tcf-4 did not influence CD97 promoter activity. Inhibition of glycogen synthase kinase (GSK)-3beta, a negative regulator of the canonical Wnt pathway, had no influence on CD97 expression levels. In summary, enhanced CD97 expression in CC cells is regulated independent of beta-catenin/Tcf-4, and is thus not a direct target of the canonical Wnt pathway.

Antigens, CD↗
PubMed2006

Particle-mediated cytokine gene therapy leads to antitumor and antimetastatic effects in mouse carcinoma models.

BACKGROUND: We investigated the effects of continuous cancer gene therapy including (antigen-presenting cell) (APC) engineering and local stimulation of the immune system. MATERIALS AND METHODS: Lewis lung carcinomas and B16 melanomas, intradermally established on C57/Bl6 mice, were shot using a gene gun every 4th day with a combination of plasmids. The first therapy group received plasmids coding the genes for interleukin (IL)-12 and IL-2. The second therapy group was treated with plasmids coding for B7.1 interferon-gamma (IFN-gamma)/IL-12 alternated by a plasmid coding IL-2. Control were mice without any therapy or treatment with the empty plasmid. RESULTS: Gene therapy led to reduced tumor sizes in the therapy groups of both models (significant for the Lewis lung carcinoma). We found an enhanced survival and reduced tumor growth rate in the therapy groups; however, the effects were not significant. IL- 12/IL-2 therapy was more effective, compared to B7.1/IFN-gamma/IL-12 and IL-2. Cytokine gene transfer let to a significantly lower metastasis rate in Lewis lung carcinoma. CONCLUSIONS: Continuous particle-mediated gene transfer is easy to handle and shows good results. Gene therapy combining the genes coding for IL-12 and IL-2 was superior to additional IFN-gamma/B7.1. APC engineering does not appear to be sufficient in these poorly antigenic tumors.

Animals↗
PubMed2006

Individual cell-based models of tumor-environment interactions: Multiple effects of CD97 on tumor invasion.

The presence of scattered tumor cells at the invading front of several carcinomas has clinical significance. These cells differ in their protein expression from cells in central tumor regions as recently shown for the EGF-TM7 receptor CD97. To understand the impact of such heterogeneity on tumor invasion, we investigated tumor cells with modified CD97 expression in vitro and in vivo. Applying an individual cell-based computer model approach, we linked specific cell properties of these cells to tumor invasion characteristics. CD97 overexpression promoted tumor growth in scid mice, stimulated single cell motility, increased proteolytic activity of matrix metalloproteinases, and secretion of chemokines in vitro in an isoform-specific manner. We demonstrated by computer simulation studies that these effects of CD97 can increase the invasion capacity of tumors. Furthermore, they can cause the appearance of scattered tumor cells at the invasion front. We identified local tumor environment interactions as triggers of these multiple capabilities. Experimentally, our simulation results are supported by the finding that CD97 expression in tumor cells is regulated by their environment. Our combined experimental-theoretical analysis provides novel insight to how variations of individual cell properties can be linked to individual patterns of tumor cell invasion.

Animals↗
PubMed2005

Coexpression of preprotachykinin A and B transcripts in the bovine corpus luteum and evidence for functional neurokinin receptor activity in luteal endothelial cells and ovarian macrophages.

Nonneuronal cell sources of tachykinins, such as substance P (SP) and neurokinin B (NKB), have been demonstrated in leukocytes, endothelial cells and endocrine cells, and may play a role in corpus luteum (CL) development. For this reason, we analyzed mRNA presence for the two tachykinin precursors together with the neurokinin-1 receptor and the neurokinin-3 receptor (NK-1R and NK-3R, preferred by SP and NKB, respectively) in bovine CL at various stages in the luteal phase. Using the RT-PCR technique, we detected coexpression for the preprotachykinin A gene (PPT-A), which encodes SP and neurokinin A (NKA), and the preprotachykinin B gene (PPT-B) for NKB in the CL at the development, secretion and regression stages. Coexpression was also noted for NK-1R and NK-3R gene transcripts. Cultures of endothelial cells (ECs) derived from bovine CL expressed NK-1R and NK-3R mRNA, as did ovarian macrophages. Agonist treatment induced a stronger intracellular calcium ([Ca2+]i) increase after activation of NK-1R compared to NK-3R, a result that we verified by calcium imaging. This is the first evidence for functional tachykinin receptor activity in luteal ECs and ovarian macrophages from bovine CL.

Animals↗
PubMed2004

N-glycosylation of CD97 within the EGF domains is crucial for epitope accessibility in normal and malignant cells as well as CD55 ligand binding.

CD97 is an EGF-TM7 receptor found on various carcinomas where expression levels correlate with dedifferentiation and tumor stage, smooth muscle cells and leukocytes. CD97 acts as an adhesion molecule by binding to its cellular ligand, CD55. In this study, we demonstrate that 2 immunodominant CD97 epitopes are not equally present in the various cell types. Differences were apparent in gastrointestinal tumors and smooth muscle cells where monoclonal antibodies (mAbs) to the first epidermal growth factor (EGF) domain (CD97(EGF)) showed a more restricted staining pattern than mAbs to the stalk region (CD97(stalk)). This discrepancy was not detectable in cultured gastrointestinal tumor cell lines. In fact, the selection of the CD97 mAb influences the result of clinical studies. Thus, we clarified the reason(s) for these differences in CD97 mAb staining on various cell types. We provide evidence that epitope accessibility for CD97(EGF) mAbs depends on N-glycosylation. Immunoprecipitation of CD97 from the Colo 205 tumor cell line revealed the established 78 and 83 kDa products, while a 52 and 57 kDa band were obtained from smooth muscle cells. N-glycosidase F reduced the size of CD97 in Colo 205 cells to 52-57 kDa. Culturing these cells with tunicamycin resulted in the same decrease in size and impaired CD97(EGF) mAb binding. As shown by site-directed mutagenesis, deletion of the N-glycosylation sites located within the EGF domains efficiently disturbed CD97(EGF) mAb immunoreactivity and, importantly, binding of CD55. In conclusion, CD97(EGF) epitope accessibility for mAbs and ligand binding is influenced by cell type-specific N-glycosylation.

Antibodies, Monoclonal↗
PubMed2004

Molecular cloning of bovine CD97: an EGF-TM7 molecule expressed as isoforms.

CD97, a cell surface molecule on immune cells with potential adhesive function, is a member of the epidermal growth factor seven-span transmembrane (EGF-TM7) family. We have cloned and characterized bovine CD97 and determined its expression in various cells and tissues. Based on sequence alignment with human genomic DNA, as well as human cDNA sequences encoding various CD97 isoforms, we predict that bovine CD97 mRNA occurs in four splice variants. The encoded CD97 isoforms (800, 751, 756, and 707 amino acids in length) contain different numbers of EGF-like modules, a stalk region, and a TM7 domain with a cytoplasmic tail. RT-PCR demonstrated expression of CD97 mRNA in leukocytes and several other tissues. In each cell type, CD97 mRNA encoding the shortest isoform, CD97 (EGF 1,2,5), was detected at the highest level, which was consistent with the cell surface expression of a 90-kDa polypeptide precipitated from lysates of peripheral blood mononuclear cells.

Amino Acid Sequence↗
PubMed2003

Detection of alternatively spliced EMR2 mRNAs in colorectal tumor cell lines but rare expression of the molecule in colorectal adenocarcinomas.

EMR2 and CD97, members of the epidermal growth factor (EGF)-TM7 family, show a very high homology. CD97, whose expression is closely related to clinical tumor stage in colorectal carcinomas, potentially functions as an adhesion molecule. Nothing is known about the presence of EMR2 in these tumors. We systematically examined the expression of EMR2 in colorectal carcinoma cell lines and adenocarcinomas. Of 18 cell lines, 10 were only slightly positive for EMR2 according to flow cytometry. Various EMR2 splice variants, including a new isoform, have been detected at the mRNA level. EMR2 expression did not correlate with in vitro migration or invasion capacity of the cell lines. Normal colorectal epithelial cells were EMR2 negative. In contrast to CD97, which is found in most colorectal adenocarcinomas, only 8 of 81 of these tumors expressed EMR2. No correlation was found between EMR2 expression and clinicopathological parameters of the tumors. In summary, a significant but low number of colorectal carcinomas are positive for EMR2, indicating different roles for this molecule and CD97 in these tumors.

Adenocarcinoma↗
PubMed2003

Induction of matrix metalloproteinase 1 gene expression is regulated by inflammation-responsive transcription factor SAF-1 in osteoarthritis.

OBJECTIVE: To identify specific transcription factors that are activated in the cartilage tissue of osteoarthritis (OA) patients and are involved in the expression of matrix metalloproteinase 1 (MMP-1). METHODS: DNase I footprint and electrophoretic mobility shift assays were performed to identify active elements of the MMP1 promoter and the transcription factors that interact with it. The relative abundance of the involved transcription factor in the cartilage was determined by immunohistochemical analysis. The role of serum amyloid A-activating factor 1 (SAF-1) in MMP-1 expression was assessed by transient transfection assay with plasmids containing either a functional or an antisense SAF-1 complementary DNA. RESULTS: A novel promoter element was detected in the human MMP1 gene, and the inflammation-responsive transcription factor SAF-1 was found to interact with it. SAF-1 activity was detected in the chondrocytes of OA cartilage, where most of the cells stained positive for SAF-1. Overexpression of SAF-1 in OA chondrocytes increased the expression of the MMP1 promoter, and interference of endogenous SAF-1 activity by antisense SAF-1 messenger RNA inhibited interleukin-1-mediated MMP1 promoter activity. CONCLUSION: The results indicate that SAF-1 is involved in the regulation of MMP1 gene expression and it is highly abundant in the articular cartilage chondrocytes of OA patients. The data also demonstrate that control of SAF-1 activity can suppress induced expression of MMP-1. Conceivably, SAF-1 could be a potential therapeutic target to control the overexpression of MMP-1 associated with the pathogenesis of OA.

Animals↗
PubMed2003

Differences between retinal and choroidal microvascular endothelial cells (MVECs) under normal and hypoxic conditions.

The morphological and functional differences between the retinal and choroidal vascular bed raise the question of whether the smallest functional unit, the microvascular endothelial cell (MVEC), also differs in its basal characteristics. Here, we examined bovine retinal and choroidal MVECs (rMVECs, cMVECs) for the presence and regulation of angiogenic mediators and their receptors, and cytokines at the mRNA level using quantitative RT-PCR and differential display. Vascular endothelial growth factor (VEGF) mRNA was expressed in both rMVECs and cMVECs. The basal and hypoxia-increased VEGF mRNA levels were significantly higher in cMVECs, which may indicate a higher capacity for autocrine stimulation in these cells. The mRNA for two VEGF receptors, Flt-1 and Flk-1, was present in rMVECs and cMVECs. Interestingly, rMVECs expressed higher Flt-1 but lower Flk-1 mRNA levels compared to cMVECs. Examining the angiopoietin (Ang)/Tie-2 system, we only detected Ang-1 mRNA at very low levels. While Ang-2 mRNA levels were high in both rMVECs and cMVECs, rMVECs expressed 2-3 times the basal and hypoxia-upregulated Ang-2 mRNA levels than did cMVECs. No difference was found in basal Tie-2 mRNA levels. rMVECs are the more potent producers of macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage CSF (GM-CSF), whereas cMVECs expressed higher RANTES mRNA levels. In our second approach - screening rMVECs and cMVECs for differentially expressed genes - we found liprin-beta1, calnexin, and sushi-repeat-containing protein, x chromosome (SRPX) mRNA in both MVEC types at varying levels. In summary, MVECs from the retinal and choroidal vascular beds showed quantitative differences in angiogenic regulator expression and in their capability to produce cytokines.

Animals↗
PubMed2003

Estrogen receptor expression and synthesis in the human internal thoracic artery.

The inhibition of vascular smooth muscle cell proliferation is mediated through two intracellular estrogen receptors (ERs), ER-alpha and ER-beta. Deletion variants of ER-alpha have been decribed for cultures of smooth muscle cells. The internal thoracic artery is frequently used as coronary artery bypass graft, yet neither has it been studied for the expression of ER subtypes nor for the synthesis of the ERs in morphologically hetergeneous smooth muscle cells. Using nested RT-PCR, we have demonstrated the mRNA for ER-alpha splicing variants in intact human internal thoracic arteries. The 7A deletion variant occurred in 8 out of 12 cases, the full-length transcript in three cases. The full-length transcript was always found for the ER-beta. Immunolocalization revealed ER-positive nuclei in the desmin-positive subset of smooth muscle cells, but not in cytokeratin (CK)-positive cells of the thickened intima. Morphological evidence is presented suggesting that ER synthesis is high in the tunica media when cell proliferation of smooth muscle cells is increased. We conclude that, in internal thoracic arteries, the 7A deletion variant of ER-a occurs in 75%, whereas the full-length transcript is found in all cases. The significance remains unclear.

Alternative Splicing↗
PubMed2002

Voltage-dependent potassium channels in cytokeratin-positive and cytokeratin-negative microvascular endothelial cells of the corpus luteum.

We have recently described cytokeratin-positive (CK(+)) and cytokeratin-negative (CK(-)) microvascular endothelial cells (MVECs) in the bovine corpus luteum. The two phenotypes show a different expression and release of adhesion molecules and cytokines. Since secretion of mediators is specifically regulated by the electrophysiological membrane parameters, this report will compare voltage-dependent potassium (K(+)) channels in the two cell types cultured under the same conditions. CK(+) and CK(-) MVECs derived from the microvascular bed of one organ differ in their K(+)-channel characteristics. In CK(-) MVECs, an inwardly rectifying K(+) current was discovered, showing the characteristics of the Kir2.1. CK(+) MVECs displayed a voltage-dependent K(+) current that activates rapidly on depolarization and inactivates very slowly, and is associated with a member of the Kv family. The mRNA for Kir2.1 was identified by RT-PCR in CK(-) and CK(+) MVECs, but there was no evidence of Kv1.4 mRNA in either of them. The function of Kir2.1 in CK(-) MVECs might be induced during cultivation, whereas CK(+) MVECs appear to be more resistant to environmental conditions and do not express an inward current.

Animals↗
PubMed2002

Microvascular endothelial cells differ in basal and hypoxia-regulated expression of angiogenic factors and their receptors.

Phenotypically and functionally different types of microvascular endothelial cells (MVECs) derived from the developing corpus luteum were isolated and characterized by our group. We investigated whether these cytokeratin-positive (CK+) and cytokeratin-negative (CK-) MVECs differed in the expression of angiogenic factors and their regulation under hypoxia. Using quantitative RT-PCR, VEGF and its receptors, Flk-1 and Flt-1, were detected in CK- MVECs. The mRNA expression of Flk-1 mRNA was 100 times as high as that of Flt-1 mRNA. CK+ MVECs expressed VEGF and Flt-1 mRNA, but were devoid of Flk-1 transcripts. No Ang-1 mRNA was demonstrated in either cell type, and Ang-2 mRNA was found only in CK- MVECs. Tie-2 mRNA was detected in both MVEC types, but levels were 150 times as high in CK- MVECs as in CK+ MVECs. mRNA of hypoxia-inducible factors Hif-1alpha and Hif-1beta was expressed in both MVEC types. After hypoxia, neither VEGF, nor Flk-1, nor Tie-2 mRNA expression was altered in either MVEC type. Flt-1 expression and Ang-2 mRNA expression were significantly increased at about 2.5-fold (P < 0.05) in CK- MVECs, but not in CK+ MVECs. Our study demonstrates the varying expression and regulation of angiogenesis-related factors and receptors in phenotypically different MVEC types.

Angiopoietin-1↗
PubMed2002

Expression and regulation of CD97 in colorectal carcinoma cell lines and tumor tissues.

The expression of CD97, a member of the EGF-TM7 family with adhesive properties, is proportional to the aggressiveness and lymph node involvement in thyroid tumors. CD97 has never been systematically investigated in other tumors. First, we examined colorectal carcinoma cell lines (n = 18) for CD97 expression and regulation. All cell lines were CD97-positive. The level of CD97 in each line correlated with migration and invasion in vitro. This result was confirmed in CD97-inducible Tet-off HT1080 cells. Transforming growth factor-beta, which inhibits proliferation in transforming growth factor-beta-sensitive LS513 and LS1034 cells, down-regulated CD97 in these cell lines. Examining CD97 during sodium butyrate-induced cell differentiation of Caco-2 cells, we could demonstrate a CD97-decreasing effect. Second, we screened 81 colorectal adenocarcinomas by immunohistology for expression of CD97. Normal colorectal epithelium is CD97-negative. Seventy-five of 81 of the carcinomas expressed CD97. The strongest staining for CD97 occurred in scattered tumor cells at the invasion front compared to cells located within solid tumor formations of the same tumor. Carcinomas with more strongly CD97-stained scattered tumor cells showed a poorer clinical stage as well as increased lymph vessel invasion compared to cases with uniform CD97 staining. In summary, CD97 expression correlates with dedifferentiation, migration, and invasion in colorectal tumor cell lines. Moreover, more strongly CD97-stained tumor cells at the invasion front of colorectal carcinomas indicate the involvement of the molecule in tumor migration and invasion.

Androstadienes↗
PubMed2002

CD97, but not its closely related EGF-TM7 family member EMR2, is expressed on gastric, pancreatic, and esophageal carcinomas.

CD97 expression is related closely to the dedifferentiation and tumor stage in thyroid carcinomas. We systematically examined the role of CD97 and its closest relative, EMR2, in normal and malignant gastric, esophageal, and pancreatic tissue. The normal tissues were EMR2-, whereas CD97 was expressed slightly in the parietal cells of gastric mucosa and in exocrine pancreatic cells. Interestingly, intralobular and interlobular pancreatic ducts were CD97+. All tumors were EMR2-. CD97 was expressed by 44 of 50 gastric, 14 of 18 pancreatic, and 10 of 13 esophageal carcinomas. Of the 44 gastric cancers, 27 showed disseminated or scattered tumor cells at the invasion front with stronger CD97 expression than tumor cells located in solid tumor formations. There was no correlation between CD97 levels in the tumors or soluble CD97 in the serum samples and the clinicopathologic features of the patients. Taken together, significant numbers of gastric, esophageal, and pancreatic carcinomas are CD97+, whereas its homolog, EMR2, does not have any role in such tumors.

Aged↗