Improved Medium for Extraction of Plasminogen Activator from Tissue

Camiolo, Sarah M.; Siuta, M. R.; Madeja, Judith M.

doi:10.1080/00327488208065678

Cited by 48 publications

(23 citation statements)

References 14 publications

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“…These histozymographic data on tPA are in agreement with the results of previous studies on tissue homogenates (de Bruin et al, 1988;Suzumiya et al, 1988;Sier et al, 1991a). Using antigen/activity immunosorbent methods together with an efficient tissue extraction procedure (Camiolo et al, 1982), we could demonstrate a clear-cut reduction in the tPA levels in adenomas compared with control and distant mucosae. The tPA antigen assay used in this study detects both free tPA and tPA complexed with inhibitor (Ranby et al, 1989).…”

Section: Discussionsupporting

confidence: 90%

Alterations in plasminogen activation correlate with epithelial cell dysplasia grading in colorectal adenomas

Protiva

Sordat²,

Chaubert³

et al. 1998

Br J Cancer

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Summary Proteases are important for neoplastic invasion but a specific role for the plasminogen activator system in the progression of colorectal epithelial dysplasia to adenomatous lesions remains unclear. Consecutive tissue cryosections of 51 adenomas, 49 distant mucosa samples and five mucosa samples from control subjects were histopathologically analysed for dysplasia grade and tissue type, urokinase plasminogen activator levels and plasminogen activator inhibitor type 1 (PAI-1) using immunosorbent methods. Plasminogen activation and urokinase-mediated proteolytic activity levels were assessed using in situ zymography. Plasminogen activation and tissue-type activator levels were lower in adenomas than in mucosae (P < 0.001). PAI-1 concentration and urokinase levels were higher in adenomas than in mucosae (P < 0.001 and P < 0.001 respectively). In adenomas, urokinase concentration increased in parallel with PAI-1, but only the urokinase levels correlated with the dysplasia grade (P < 0.01). Thus, the alterations in plasminogen activation correlated with epithelial cell dysplasia grading. In the mucosa to adenoma transition, a marked decrease in tissue-type plasminogen activator occurred. In adenomas, this decrease was accompanied by a concomitant increase in urokinase and PAI-1. The urokinase level only continued to rise in parallel with the dysplasia grade. Resulting protease-antiprotease imbalance in high-grade dysplasia may represent the phenotypic change associated with malignant transformation and invasive behaviour.Keywords: colorectal adenoma; epithelial cell dysplasia; plasminogen activator inhibitor type 1; tissue-type plasminogen activator; urokinase-type plasminogen activator A series of phenotypic changes are associated with the malignant conversion of the colorectal epithelium. The grade of epithelial cell dysplasia is a hallmark of malignant potential in the process known as the adenoma-carcinoma sequence (Muto et al, 1975;O'Brien et al, 1990;Hamilton, 1992). While the morphological changes taking place during the development and progression of colorectal dysplasia can readily be detected by histopathology, the proteolytic alterations underlying these multiple events remain unclear.It has been shown that components of the plasminogen activation system are key participants in the regulation of extracellular matrix turnover during cell migration, tissue modelling and malignant invasion (Konishi et al, 1982;Vassalli et al, 1991;Blasi, 1993). Previous studies have shown that tissue extracts from colorectal carcinomas have increased levels of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitors (PAI) and decreased levels of tissue-type plasminogen activator (tPA) compared with morphologically normal adjacent mucosa (Gelister et al, 1986;Sier et al, 1991a). In adenomas, the levels of activators and inhibitors appear to be between those found in normal mucosa and those found in carcinoma (Gelister et al, 1986;Bruin et al, 1987;Sim et al, 1988;Suzumiya et al, 1988;Sier et al, ...

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Section: Discussionsupporting

confidence: 90%

Alterations in plasminogen activation correlate with epithelial cell dysplasia grading in colorectal adenomas

Protiva

Sordat²,

Chaubert³

et al. 1998

Br J Cancer

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“…Dilution of cytosols was performed applying dilution buffers recommended for that particular assay procedure. The following extraction buffers were used for pellet extraction: (1) Jänicke buffer; 20 mM Tris-HCl, 125 mM NaCl containing 1% Triton X-100, pH 8.5 [20]; and (2) Camiolo buffer: 75 mM potassium acetate, 0.3 M NaCl, 0.1 M L-arginine, 10 mM K2EDTA, 0.25% Triton X-100, pH 4.2 [21].…”

Section: Samplesmentioning

confidence: 99%

“…One pellet was extracted with a buffer previously described by Jänicke and colleagues [20] which contains the non-ionic detergent Triton X-100 (pH 8.5). The other pellet was extracted with a buffer described by Camiolo and coworkers [21], This latter buffer has a high ionic strength and a low pH (pH 4.2). Both extracts were diluted to various extents not with the extraction buffers but with the appropriate dilution buffers belonging to the particular assay procedure.…”

Section: Upa In Tissu E Pellet Extractsmentioning

confidence: 99%

Immunoassays (ELISA) of urokinase-type plasminogen activator (uPA): Report of an EORTC/BIOMED-1 Workshop

Benraad

Geurts-Moespot

Grøndahl-Hansen

et al. 1996

European Journal of Cancer

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“…In brief, the frozen tissue was mechanically pulverized with a dry ice-cold powder pistol. Tissue powder was suspended in ice-cold acetate-detergent low pH extraction buffer (33). Subsequently the solution was centrifuged at 105,000 ϫ g for 1 h at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Measurement of the Uncomplexed Fraction of Tissue Inhibitor of Metalloproteinases-1 in the Prognostic Evaluation of Primary Breast Cancer Patients*

Würtz¹,

Christensen

Schrohl³

et al. 2005

Molecular & Cellular Proteomics

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Several studies have demonstrated an association between high tumor tissue levels of total tissue inhibitor of metalloproteinases-1 (TIMP-1) and a poor prognosis of primary breast cancer patients. In the present study we investigated whether measurements of the uncomplexed fraction of TIMP-1 added prognostic information to that already obtained from total TIMP-1. We measured the uncomplexed fraction of TIMP-1, using a thoroughly validated ELISA specific for this fraction, in 341 tumor tissue extracts obtained from patients with primary breast cancer. These measurements were related to previously performed measurements of total TIMP-1 as well as to patient outcome. The observation time was 8.3 years (range, 7.3-11.3 years). During this period 136 patients died, and 153 patients experienced recurrence of disease. Cox regression analysis of recurrence-free survival (RFS) suggested that a score based on both uncomplexed and total TIMP-1, reflecting the tumor level of TIMP-1/MMP complexes, would be a more precise estimate of prognosis than total TIMP-1 alone. Univariate survival analysis showed a highly significant relationship between high values of the score and poor outcomes for RFS (p ‫؍‬ 0.0002; hazard ratio ‫؍‬ 2.7; 95% confidence interval, 1.5-4.8). Similar results were found for overall survival (p ‫؍‬ 0.0001; hazard ratio ‫؍‬ 3.3; 95% confidence interval, 1.8 -6.3). Multivariate analysis of RFS and overall survival demonstrated that the score was significant including the classical prognostic factors used in breast cancer (p < 0.0001). The present study raises the hypothesis that it is the tumor level of TIMP-1/MMP complexes (i.e. activated matrix metalloproteinases) rather than TIMP-1 itself that determines prognosis, supporting the use of the combined score and not only total TIMP-1 in stratification of breast cancer patients. Molecular & Cellular Proteomics 4:483-491, 2005.

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Improved Medium for Extraction of Plasminogen Activator from Tissue

Cited by 48 publications

References 14 publications

Alterations in plasminogen activation correlate with epithelial cell dysplasia grading in colorectal adenomas

Alterations in plasminogen activation correlate with epithelial cell dysplasia grading in colorectal adenomas

Immunoassays (ELISA) of urokinase-type plasminogen activator (uPA): Report of an EORTC/BIOMED-1 Workshop

Measurement of the Uncomplexed Fraction of Tissue Inhibitor of Metalloproteinases-1 in the Prognostic Evaluation of Primary Breast Cancer Patients*

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