Immunohistochemical quantification of lymph vessels, VEGF-C and VEGF receptor 3 in human sarcomas.
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Humans↗
Biomedical subjects
N Wernert
Publications and source records attributed to N Wernert.
[New biological concepts in cardiovascular pathology].
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Cardiovascular Diseases↗
[Lymph vascularity and lymph node metastases on PET and PET-CT: immunohistological and clinical observations].
Sarcomatous malignancies only rarely develop regional lymph node metastases: about 2.7% of our evaluated cases. In this paper we provide evidence supporting a new hypothesis that two entirely separate lymph vascular systems exist in humans. One system (LGS I) exists in close proximity to the epithelium and drains into regional lymph nodes. Only sarcomas that originate in the epithelium or its immediate proximity are able to form regional lymph node metastases. The vast majority of sarcomatous malignancies (97.4% of cases) do not give rise to lymph node metastases, since they originate in proximity to a second, more deeply localized lymph node system (LGS II) in the mesenchymally derived tissues of the body. This second system has no connection to regional lymph nodes. Supporting evidence is provided by experience in the operative treatment of extremity lymphedema, PET-CT examinations, radionuclear lymphography, and scientific investigations using antibodies specifically directed at the elements of the lymph vascular system.
Epithelium↗
GSTP1 hypermethylation as a molecular marker in the diagnosis of prostatic cancer: is there a correlation with clinical stage, Gleason grade, PSA value or age?
OBJECTIVES: Epigenetic events such as promoter hypermethylation have been implicated in prostate carcinogenesis. We present a real-time, methylation specific protocol to detect hypermethylation in the promoter region of the GSTP1 gene in benign hyperplasia and adenocarcinoma of the prostate. METHODS: In our preliminary study, 31 prostate cancer and 5 benign prostatic hyperplasia (BPH) tissue samples were analyzed. Genomic DNA was isolated from formalin-fixed and paraffin-embedded specimens and subjected to sodium bisulfite modification, followed by real-time, methylation specific PCR. Patients with prostatic cancer were also subdivided according to their Gleason score, PSA, age and TNM Staging. Prostate cancer cell lines (LNCaP, DU145, PC3) and a BPH cell line (BPH-1) were also tested as controls. RESULTS: GSTP1 promotor hypermethylation was detected in 28 of the 31 prostate cancer cases (90.3%) and none of the five (0%) BPH cases. Statistical analysis did not reveal a significant correlation between GSTP1 hypermethylation and Gleason score, PSA, age or TNM staging. All prostate cancer cell lines were testes positive for GSTP1 promotor hypermethylation, whereas the BPH cell line (BPH-1) was tested negative. CONCLUSION: GSTP1 promotor hypermethylation occurs during carcinogenesis and is considered to be a major event of prostate carcinogenesis. Our data support this thesis and shows that GSTP1 hypermethylation reliably distinguishes between prostate cancer and BPH . Although it is not yet clear at what time during carcinogenesis hypermethylation of the GSTP1 promotor occurs it seems to provide valuable information for prostate cancer screening and diagnosis. Larger studies are underway to determine the potential role for GSTP1 hypermethylation in clinical settings.
Adenocarcinoma↗
[Molecular biological and immunohistochemical analysis of tp53 in human ameloblastomas].
AIM: Ameloblastoma is the most frequent epithelial tumor of the jaws. The spread is locally invasive and it tends to recur. Malignant transformation and occurrence of metastases has been described. Immunohistochemical analysis shows an enhanced expression of P53 protein in ameloblastomas. Mutation of the tp53 tumor suppressor gene was verified in several human tumors. In this study histological sections were analyzed for the expression of P53 protein and the tp53 gene was examined for mutations. MATERIAL AND METHODS: Tumor DNA from 29 patients with an ameloblastoma was examined for mutations in exons 5 to 8 of the tp53 tumor suppressor gene using PCR, SSCP,- and sequential analysis. Histological sections of the tumors were analyzed immunohistochemically for an overexpression of P53 protein. RESULTS: Two tp53 mutations (6.9%) in ameloblastomas were verified for the first time. In all 58.6% of the tumors showed an immunoreactivity for P53 protein. There was a statistically significant positive correlation (Fisher's exact test p<0.0148) between an increased number of P53-positive tumor cells and the appearance of recurrence. DISCUSSION: In the face of the uncertain postoperative behavior of ameloblastomas, the immunohistochemical verification of more than 10% P53-positive tumor cells may give a prognostic indication for a tendency to recurrence.
Aged↗
The Ets-1 transcription factor is involved in the development and invasion of malignant melanoma.
The Ets-1 transcription factor plays a role in tumor vascularization and invasion by regulating expression of matrix-degrading proteases in endothelial cells and fibroblasts in the tumor stroma. During early embryogenesis, Ets-1 is expressed in migrating neural crest cells from which melanocytes arise. In the present study, we analyzed Ets-1 expression in various melanocytic lesions and investigated its functional importance in malignant melanomas. We found that Ets-1 was upregulated both in vivo and in vitro in malignant melanoma, compared to benign melanocytic lesions and to primary melanocytes. Assessment of DNA-binding and transactivation assays documented a strong Ets activity in melanoma cells. Using an antisense strategy, the expression and activity of Ets-1 were reduced in the melanoma cell line Mel Im. This correlated with a diminished expression of several Ets-1 target genes known to be involved in invasion, such as MMP1, MMP3, uPA and integrin beta3. In line with these findings, the invasive potential of the melanoma cells measured in a Boyden Chamber model was reduced up to 60% after Ets-1 blockade. This can be attributed to the role of Ets-1 in transcriptional regulation of factors involved in invasion of melanoma cells. We conclude that over-expression of Ets-1 during melanoma development contributes to the malignant phenotype.
DNA, Neoplasm↗
[Laser microdissection in the molecular oncology of prostate cancer].
Nearly all diseases, including prostate cancer (PCA), occur in mixed tissues with different cell types interconnected by multiple interactions. Laser microdissection permits a separate analysis of specific cell types necessary to understand tumorigenesis. Microdissection can be combined with different molecular methods for analyses at the levels of the genome, the transcriptome or the proteome. With respect to the molecular pathogenesis of PCA, normal glands can be compared to preneoplasias, and these in turn to the carcinoma. Different malignancy grades, as well as intra- and extraprostatic tumor parts, can be specifically analysed and molecular markers of aggressiveness can be identified. The molecular signatures obtained provide the basis for functional studies. New prognostic markers and therapeutic targets can be expected from such approaches in the near future. A far reaching goal is the computer representation of multiple molecular components and their interactions, "E-cell in cyberspace", in which prognostic behaviour and therapeutic responsiveness can be approximately predicted.
Animals↗
A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction.
Quantification of nucleic acids, especially of mRNA, is increasingly important in biomedical research. The recently developed quantitative real-time polymerase chain reaction (PCR) - a highly sensitive technology for the rapid, accurate and reproducible quantification of gene expression - offers major advantages over conventional quantitative PCR. Transcript quantification is performed in the exponential phase of the PCR reaction through extrapolation of fluorescence signals from a standard calibration curve which represents the initial copy number for a given fluorescence signal. We have developed a method for gene transcript quantification which is based on a LightCycler - assisted real-time PCR in combination with a simple and rapid approach for the construction of external cRNA standards with identical gene sequences as the target gene. Synthesis of cRNAs was performed by in vitro transcription with T7 RNA polymerase followed by reverse transcription and real-time PCR. We applied this approach for transcript quantification of eukaryotic initiation factor 3 p110 (EIF3S8) mRNA in normal testicular tissue. We also present a rapid and simple strategy for the construction of cRNA standards for use in real-time PCR.
Adult↗
[From SKY, chips and proteomics. Molecular medicine in the time of high technology].
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Biomedical Technology↗
The Ets-1 transcription factor is up-regulated together with MMP 1 and MMP 9 in the stroma of pre-invasive breast cancer.
The first steps of stroma generation are of pivotal importance for carcinogenesis because at this stage are initiated both angiogenesis, the prerequisite for continuous tumour growth, and the proliferation of stromal fibroblasts. These developments contribute to the onset of tumour invasion by secreting several matrix-degrading proteases. Both angiogenesis and the production of proteases are tightly controlled at several levels; of significant importance is transcription. The Ets-1 transcription factor transactivates several genes encoding matrix-degrading proteases and is thought to be involved in both tumour vascularization and invasion. This study therefore investigated, by in situ hybridization and immunohistochemistry, the expression of Ets-1 and of two of its target genes, encoding matrix metalloproteinase (MMP) 1 and MMP 9, in order to demonstrate a topographical in vivo correlation between the expression of these three genes during breast cancer formation. All three genes were first expressed within both endothelial cells and stromal fibroblasts during the onset of stroma generation around intraductal and intralobular in situ carcinomas and they were significantly up-regulated in the stroma of invasive ductal and lobular cancers. The results of this study further support the suggested in vivo role of Ets-1 for both angiogenesis and tumour invasion, via matrix-degrading proteases which are already expressed during the early stages of breast carcinogenesis.
Breast Neoplasms↗
Ectopic thymus presenting as a submandibular swelling.
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Biopsy↗
Downregulation of clusterin expression in testicular germ cell tumours.
OBJECTIVES: Clusterin is implicated in many biological processes including cell adhesion, apoptosis and transformation. Clusterin expression was demonstrated during sperm maturation and is overexpressed in different malignancies including breast and prostate carcinomas and anaplastic large-cell lymphomas. METHODS: The aim of this study was to determine the expression of clusterin in a series of different germ cell tumours by immunohistochemistry. Twenty-two seminomas, 27 embryonal carcinomas, 22 mature and immature teratomas, 8 yolk sac tumours and 1 chorion carcinoma as well as 30 normal testes were analysed using a monoclonal antibody. RESULTS: In normal testes strong signals were seen in the maturing germ cells and in Leydig cells. In most tumours examined no clusterin expression was detected (84%). Only a focal weak expression was seen in some teratomas (32%), embryonal carcinomas (15%) and yolk sac tumours (25%). CONCLUSIONS: Our results suggest that clusterin is associated with normal germ cell development and that the loss of clusterin expression might play a role in the malignant transformation of germ cells.
Biomarkers↗
Invasive properties of serous human epithelial ovarian tumors are related to Ets-1, MMP-1 and MMP-9 expression.
The invasive potential of serous epithelial ovarian tumors is the main factor determining their biological behaviour. In contrast to invasive serous ovarian carcinomas serous borderline tumors generally present without stromal invasion and without or non-invading peritoneal implants. Little is known about the reasons underlying these differences. In the present study we found that two matrix-degrading metalloproteinases, collagenases 1 and 4 (MMPs 1 and 9), as well as the Ets-1 transcription factor are expressed at very low levels in serous benign cystadenomas, upregulated in the fibroblastic stroma, but not in the epithelium of borderline tumors and most strongly expressed in both stromal and epithelial tumor cells of serous invasive carcinomas. Since expression of Ets-1 and of MMPs 1 and 9 are topographically related, a transcriptional regulation of both proteases by Ets-1 is suggested. Upregulation of MMPs 1 and 9 within fibroblastic stromal cells of borderline tumors might be related to matrix remodelling and additional expression of both enzymes by the neoplastic cells of invasive carcinomas could then allow invasive propagation. The different expression patterns might supports the view, that no transition of serous borderline tumors into invasive carcinomas occurs.
3T3 Cells↗
Eukaryotic initiation factor 3 p110 mRNA is overexpressed in testicular seminomas.
Testicular germ cell tumors are important neoplasms and seminoma accounts for 40 to 50% of these tumors. Little is known concerning the molecular events underlying the development of these malignancies. In the present study we used a modified differential display approach to compare gene expression between seminoma and normal testicular parenchyma, both of which are mixed tissues. We first analyzed mRNA (cDNA) expression by differential display and then directly used differentially expressed cDNAs for the synthesis of radiolabeled riboprobes to attribute differential expression to specific cell types in tissue sections by in situ hybridization. Using this approach along with real-time quantitative reverse transcriptase-polymerase chain reaction analysis we found an overexpression of eukaryotic initiation factor 3 p110 mRNA (EIF3S8) in seminoma cells compared to normal germ cells of testicular tubules. The elF3-p110 subunit may promote seminoma development by generally increasing translation leading to enhanced cellular growth and division rates.
Adult↗
Presence of genetic alterations in microdissected stroma of human colon and breast cancers.
We show by laser-assisted microdissection that frequent genetic alterations in non-hereditary invasive human colon and breast cancers (loss of heterozygosity and TP53 mutations) occur not only in the neoplastic epithelial cells, but also in the adjacent fibroblastic tumor stroma and that both components can share clonal features. Tumor cell-mesenchyme transitions are among the possible explanations for these findings.
Breast Neoplasms↗
Inhibition of Angiogenesis In Vivo by ets-1 Antisense Oligonucleotides-Inhibition of Ets-1 Transcription Factor Expression by the Antibiotic Fumagillin.
The inhibition of angiogenesis in vivo as a result of the inhibition of Ets-1 transcription factor expression by the ets-1 phosphorothioate antisense oligodeoxynucleotide 5'-AGATCGACGGCCGCCTTCAT-3' has been proven by experiments with chicken embryos. Thus, participation of the Ets-1 transcription factor in the formation of new blood vessels in vivo has been demonstrated. Furthermore, it is shown that the angiostatic effect of the fungal metabolite and angiogenesis inhibitor fumagillin is mainly a result of its ability to inhibit Ets-1 expression.
Journal Article↗
Expression of the Ets-1 transcription factor in human astrocytomas is associated with Fms-like tyrosine kinase-1 (Flt-1)/vascular endothelial growth factor receptor-1 synthesis and neoangiogenesis.
Marked neovascularization and vascular endothelial proliferation are characteristic features of malignant gliomas. Vascular endothelial growth factor (VEGF), an angiogenic protein secreted by glioma cells, appears to play a crucial role for induction of neoangiogenesis. The VEGF receptors fms-like tyrosine kinase-1 (Flt-1)/VEGFR-1 and kinase insert domain-containing receptor (KDR)/ VEGFR-2 are up-regulated on the surface of endothelial cells (ECs) in gliomas. Both receptor genes contain an Ets-responsible element in their promoters. The proto-oncogene ets-1 encodes a transcription factor that has been associated with blood vessel formation in vivo under physiological and pathophysiological conditions including tumor neovascularization. Ets-1 is induced by VEGF in cultured ECs. In vitro data also point to a role of Ets-1 as a transcriptional activator of Flt-1. These properties prompted us to investigate Ets-1 expression in 32 human astroglial tumors of WHO grades I-IV and to correlate the data with the expression pattern of VEGF, Flt-1, and KDR. By in situ hybridization, high ets-1 mRNA levels were found in the glioma microvasculature with particularly prominent signals in glomeruloid vascular endothelial proliferations of glioblastomas (WHO grade IV). Semiquantitative reverse transcription-PCR identified the full-length ets-1 transcript but none of three known splice variants encoding isoforms with different functional domains. Immunohistochemical staining demonstrated Ets-1 protein preferentially in the nucleus of those ECs with an epithelioid morphology consistent with an activated state, whereas quiescent flat-shaped ECs predominantly displayed cytosolic immunoreactivity. This observation proposes nuclear translocation of Ets-1 during neoangiogenesis. VEGF synthesis by glioma cells was accompanied by Ets-1 expression in adjacent microvascular ECs. Furthermore, a highly significant correlation was observed between Ets-1 and Flt-1 (but not KDR) expression in ECs of the glioma microvasculature. Our data suggest that VEGF secreted by glioma cells induces Ets-1 in adjacent microvascular ECs, which subsequently transactivates the VEGF receptor Flt-1. This cascade may crucially promote neoangiogenesis in human gliomas.
Adult↗
Cultured endothelial cells from human arteriovenous malformations have defective growth regulation.
Vascular malformations are frequent in newborns, and they persist throughout life, which differentiates them from vascular tumors (eg, hemangiomas). Arteriovenous malformations are high-flow vascular malformations. They are considered nonmalignant but can expand and become a significant clinical risk when extensive. To characterize endothelial cells from arteriovenous malformations (AMEC), we cultured cells obtained from surgical specimens and studied their properties. After selection, the cells that grew out from explants had phenotypic and antigenic features (platelet endothelial cell adhesion molecule, von Willebrand factor) of human endothelial cells. Their spontaneous proliferation rate was higher (1.8 to 6.4 times) than that of human umbilical vein, arterial, or microvascular endothelial cells. The proliferation rate of AMEC was not sensitive to the inhibitory activity of various cytokines (interleukin-1beta, tumor necrosis factor-alpha, transforming growth factor-beta, Interferon-gamma). In basal conditions, intercellular adhesion molecule (ICAM-1) was detected at a higher level of expression (6- to 10-fold) on AMEC, but these cells failed to express E-selectin or the vascular cell adhesion molecule (VCAM-1) after cytokine stimulation. Expression of c-ets-1 proto-oncogene was shown by in situ hybridization. The low response to cytokines, the higher propensity to proliferate, and the ets-1 expression suggest that AMEC have a defective regulation of proliferation that may be due to a reduced apoptotic process.
Adolescent↗